Theranostics 2020, Vol. 10, Issue 12 5435

Ivyspring International Publisher Theranostics 2020; 10(12): 5435-5488. doi: 10.7150/thno.40068 Review Graphene and other 2D materials: a multidisciplinary analysis to uncover the hidden potential as cancer theranostics Laura Fusco1,2,3,#, Arianna Gazzi1,2,#, Guotao Peng4,#, Yuyoung Shin5, Sandra Vranic6, Davide Bedognetti3, Flavia Vitale7, Acelya Yilmazer8,9, Xinliang Feng10, Bengt Fadeel4,, Cinzia Casiraghi5,, Lucia Gemma Delogu2,10,11,

1. Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy. 2. Fondazione Istituto di Ricerca Pediatrica, Città della Speranza, Padua, Italy. 3. Cancer Program, Sidra Medicine, Doha, Qatar. 4. Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 5. Department of Chemistry, University of Manchester, Manchester, UK. 6. Nanomedicine Lab, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK. 7. Department of Neurology, Bioengineering, Physical Medicine & Rehabilitation, Center for Neuroengineering and Therapeutics, University of Pennsylvania, Philadelphia, USA; Center for Neurotrauma, Neurodegeneration, and Restoration, Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, USA. 8. Department of Biomedical Engineering, Ankara University, Ankara, Turkey. 9. Stem Cell Institute, Ankara University, Ankara, Turkey. 10. Faculty of Chemistry and Food Chemistry, School of Science, Technische Universität Dresden, Dresden, Germany. 11. Department of Biomedical Sciences, University of Padua, Padua, Italy.

#These authors contributed equally to the present work.

 Corresponding author: [email protected]; [email protected]; [email protected]

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2019.09.07; Accepted: 2019.12.23; Published: 2020.04.07

Abstract Cancer represents one of the main causes of death in the world; hence the development of more specific approaches for its diagnosis and treatment is urgently needed in clinical practice. Here we aim at providing a comprehensive review on the use of 2-dimensional materials (2DMs) in cancer theranostics. In particular, we focus on graphene-related materials (GRMs), graphene hybrids, and graphdiyne (GDY), as well as other emerging 2DMs, such as MXene, tungsten disulfide (WS2), molybdenum disulfide (MoS2), hexagonal boron nitride (h-BN), black phosphorus (BP), silicene, antimonene (AM), germanene, biotite (black mica), metal organic frameworks (MOFs), and others. The results reported in the scientific literature in the last ten years (>200 papers) are dissected here with respect to the wide variety of combinations of imaging methodologies and therapeutic approaches, including drug/gene delivery, photothermal/photodynamic therapy, sonodynamic therapy, and immunotherapy. We provide a unique multidisciplinary approach in discussing the literature, which also includes a detailed section on the characterization methods used to analyze the material properties, highlighting the merits and limitations of the different approaches. The aim of this review is to show the strong potential of 2DMs for use as cancer theranostics, as well as to highlight issues that prevent the clinical translation of these materials. Overall, we hope to shed light on the hidden potential of the vast panorama of new and emerging 2DMs as clinical cancer theranostics.

Key words: 2D materials, cancer theranostics, future perspectives, graphene, nanomedicine.

1. Introduction Graphene, a single layer of graphite, is (2DM), due to its outstanding properties that can be undoubtedly the most famous 2-dimensional material exploited in various applications, ranging from

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5436 electronics to composites and biomedical applications perspective. Furthermore, these materials are suitable [1–3]. Moreover, an entire family of graphene-related for multiple functionalizations, which is fundamental materials (GRMs), each with its own properties, is for the treatment and diagnosis of cancer, or both available – ranging from chemical derivatives of (theranostics), as we will discuss in more detail in this graphene, such as graphene oxide (GO), review. nano-graphene oxide (NGO), and reduced graphene In the present review, the state-of-the-art of oxide (rGO), to new 2DMs, such as graphdiyne graphene and 2DMs in cancer theranostics is (GDY). Furthermore, graphene hybrids can be easily presented by depicting the four main scenarios of obtained by combining single or few-layer graphene nano-based approaches in cancer, namely: i) imaging (FLG) or other GRMs with different types of methods, ii) drug and gene delivery, iii) photothermal nanomaterials (e.g., quantum dots (QDs), metallic and therapy (PTT), and iv) photodynamic therapy (PDT). semiconducting nanomaterials, etc.). Although i) Imaging in cancer. Cancer imaging is graphene quantum dots (GQDs) are not necessarily indispensable not only to enable the early detection of 2D, they have been used for biomedical applications, cancer, but also to determine the precise tumor thus these are also included in this review for sake of location and stage, as well as to direct therapy and completeness. In addition, the family of 2DMs is very check for cancer recurrence after the treatment. large, and other materials have started to be used in Various imaging techniques, including positron nanomedicine, for instance the family of transition emission tomography (PET), X-ray computed metal dichalcogenides (TMDs), such as tungstenum tomography (CT), magnetic resonance imaging (MRI) disulphide (WS2) and molybdenum disulfide (MoS2), and ultrasonography, are available for clinical hexagonal boron nitride (h-BN), nitrides and applications, while other methodologies that are carbonitrides (MXenes), black phosphorus (BP) attracting the interest of the biomedical research nanosheets, silicene, antimonene (AM), germanene, community, such as fluorescence molecular biotite, metal organic frameworks (MOF), and layered tomography (FMT), are still at the preclinical stage double hydroxide (LDH). [11–14]. Since each technology has its unique One of the main goals in modern oncology strengths and limitations, hybrid imaging platforms, overlaps with that of nanomedicine: the development such as PET-CT, FMT-CT, FMT-MRI, and PET-MRI of alternative approaches through the combination of are being developed to improve data reconstruction two or more forms of treatments and diagnostic and visualization. New nanotechnology-based agents techniques. To achieve this aim, advanced may be beneficial when it comes to proposing new nanomaterials are extensively considered as they can imaging tools that will help overcome the common be used as imaging components for cancer issues of current agents (e.g., toxicity, such as the one detection/visualization with customized therapeutic related to X-ray contrast agent, and lack of specificity, agents, as well as vectors for controlled-release common to both X-ray and MRI contrast agents, and mechanisms and targeting strategies, making increased hypersensitivity following chemotherapy). nanomaterials promising nanotheranostic tools [4]. Some 2DMs show strong light-matter interaction According to the traditional definition, a effects, such as fluorescence, making them an ideal theranostic agent consists of a therapeutic and base to build experimental theranostic tools [15]. imaging (aimed at diagnosis) component combined Moreover, the 2-dimensionality enables easy load of within a single formulation. A more recent view specific nanomaterials and molecules, which provide expanded the definition of theranostics by including complementary properties to the selected 2DMs as a tools where imaging is performed to aid or guide the vector (e.g., electrochemical properties, magnetic therapy and not necessarily to perform a diagnosis [5– functions, fluorescence, radioactivity, etc.), allowing 9]. Theranostic nanomedicines offer the opportunity to enhance and expand their potential use in oncology to combine into a single nanoplatform multiple for imaging and diagnosis. imaging methodologies and therapeutic functions, ii) Drug and gene delivery. Since 1995, when such as passive and active targeting for cancer Doxil entered into the clinic for the delivery of the therapy (theranostics for drug delivery), and potent aromatic anticancer agent doxorubicin (DOX), stimuli-responsive drug release (e.g., theranostics for several nanotools have been approved for drug temperature- and pH-dependent therapy) [10]. delivery, and the list of nanodrugs in clinical trials is In general, 2DMs shows outstanding properties, still growing [16]. The ability to multifunctionalize such as remarkable light-weightness and flexibility, nanomaterials and nanoparticles (NPs), the high drug high surface-to-volume ratio, near-infrared (NIR) loading capacity, the possibility to reduce the drug light absorption, and characteristic Raman spectrum, exposure in the undesired tissue, and the improved which make them very attractive from a biological drug solubility represent the main advantages of

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5437 using nanosystems in cancer treatment. None of the established as a clinical treatment modality for cancer nanotherapeutics approved so far, mainly various diseases, including cancer, and particularly based on liposomal, silicon, gold, and iron particles, for the treatment of superficial tumors (e.g., possess the same physical/chemical properties of esophagus, bladder, and melanoma) [32,33]. Similar graphene or other 2DMs [17,18]. Systems can be to PTT, also PDT mechanism relies on the presence of designed to localize at the target disease site, which a PS; however, in this case, its activation is obtained would allow the early detection of tumors. An ideal through the illumination with visible light, instead of material, in this case, may be able to achieve the local heat application. The combined action of the molecular targeting that can be imaged during its excited triplet photosensitizer and molecular oxygen 1 circulation in the body. Upon reaching its destination, results in the formation of singlet oxygen ( O2), which it may have targeting moieties that associate with is thought to be the main mediator of cellular death cell-surface receptors, internalize into the cytosol, induced by PDT. reach an intracellular target if necessary, and release To date, the most studied nanotheranostics for the active therapeutic [7,18,19]. The effectiveness of a PTT and PDT involve organic NPs, such as polymeric drug conjugate is related to its ability to improve NPs or liposomes and inorganic NPs including therapeutic index relative to free drug, generally by quantum dots (QDs) and silica NPs. Owing to their reducing toxicity and enhancing efficacy. Historically, unique properties of high optical absorption capacity a panel of nanosystems, including liposomes, metal in the NIR region and photothermal conversion, NPs, and carbon-based materials, were modified via carbon-based nanomaterials represent ideal non-covalent or covalent modification in order to candidates for PTT [34–37]. The ability of some 2DMs deliver chemotherapeutic agents, such as DOX and to respond to light is exploited in optical therapies, paclitaxel, or agents for gene therapy (e.g., short including PTT and PDT. Finally, thanks to the interfering RNA, siRNA) [20–23], in combination with adsorption onto their surface of specific molecules imaging agents (encapsulated or intrinsic) to the and NPs, these nanotools can be endowed with tumor site [24–26]. additional magnetic, radioactive, or electrochemical iii) Photothermal therapy (PTT) is a type of properties. localized treatment that relies on the presence of an optical absorbing agent, also known as 2. Further considerations before exploring photosensitizer (PS), which can absorb energy and the potential of 2DMs as cancer convert it into heat upon stimulation by an theranostics electromagnetic radiation, such as radiofrequency, Based on the needs in oncology, and the microwaves or NIR irradiation [27]. When compared available imaging techniques and innovative to conventional radiotherapy or chemotherapy [28], therapeutic treatments, the 2DMs family should the key advantages of PTT include the capability of demonstrate a list of properties before being deep tissue penetration and minimal nonselective cell introduced into the clinic: death in the surrounding healthy tissue [27]. In the a) Lack of toxicity/acceptable biocompatibility biological environment, overheating of the PS may (i.e., are the 2DMs able to escape immune lead to hyperthermia that may cause several recognition?); hazardous effects, such as protein denaturation and b) Selective toxicity to cancer cells (i.e., are 2DMs aggregation, evaporation of cytosol, cell lysis, selective to all cancer cells or only specific types?) and apoptosis, and necrosis. An ideal PS should possess a personalized medicine (i.e., are different 2DMs high selectivity towards the target tissue, together needed for different patients?); with large absorption cross-sections for optical c) Appropriate biodistribution (e.g., in relation to wavelengths, low toxicity, easy functionalization the type of route of injection, are 2DMs degraded capability, and high solubility in biocompatible and/or excreted?); solutions [29]. PTT has been widely used in d) When designed for drug/gene delivery, nanomedicine alone or in combination with other 2DMs must be able to protect the conjugated drugs therapies and imaging modalities, such as gene from degradation, facilitate their solubilization, therapy [30] and photoacoustic imaging (PAI) [31], sustain their release, and selectively target cancer and many are the types of nanomaterials investigated cells; in this direction such as gold nanoparticles and e) When designed for PTT, 2DMs must be stable PEGylated silica-cored Au nanoshells, which are the and possess large absorption cross-sections at the first photothermal nanoparticles that have advanced specific excitation wavelengths; into clinical trials. f) When designed for PDT, the 2DM must be able iv) Photodynamic therapy (PDT) is now well to act as a PS, being activated by light of a specific

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5438 wavelength and producing a form of singlet oxygen 3. Literature review: looking back at ten that kills nearby cells. years of 2DMs theranostic research In addition to the above points, 2DMs performance in different application must also be A systematic and critical review of the literature benchmarked against other particles and materials in on graphene, GRMs, graphene hybrids, and new clinical trials (are they capable of competing with emerging 2DMs, studied in biomedicine as nanotools other particles already at the stage of clinical trials?). for cancer theranostic applications, was performed In the present review, the theranostic use of according to the Preferred Reporting Items for 2DMs is dissected from a different perspective Systematic Reviews and Meta-Analyses (PRISMA) compared to the recent reviews published on this guidelines. topic so far [9,38–46]. First, we discuss the studies The search has been carried out using different carried out, from an oncological and biological point electronic databases as data sources (PubMed, Scopus, of view, highlighting the multifunctional complexity and ToxLine); the following predetermined of the developed 2DMs in terms of different types of keywords, related with the selected nanomaterials imaging approaches, therapeutic techniques, and their application in cancer theranostics, were conjugated drugs/targeting moieties, as well as the used in several different combinations: graphene, relative targeted cancer. Our review also describes the GRMs, GO, rGO, GQDs, NGO, GDY, MXene, WS2, material characterizations performed – as the MoS2, hBN, BP, silicene, AM, germanene, biotite, MOF properties of 2DMs change with functionalization and or LDH and theranostics, 2DMs, drug delivery and processing, a comprehensive characterization is cancer, gene delivery and cancer, imaging and cancer, necessary to ensure reproducibility and, therefore, PTT and cancer, PDT and cancer, cancer diagnosis, envisage the use of 2DMs in nanomedicine. The first cancer therapy, theranostic nanomaterials, theranostic part of this review focuses on the most used 2DMs nanosystems, or theranostic nanoplatforms. As an (i.e., graphene, and its chemical derivatives and additional tool, the research was extended by hybrids) [47], and the multitude of characteristics consulting the literature of relevant reviews and exploited for therapy and diagnosis of cancer, included studies in the field of nanotechnologies and highlighting multiple combined purposes: imaging, theranostics. The list of reported studies includes all drug/gene delivery, PTT, and PDT. In contrast to the retrieved publications from 2008 to July 2019. The previous reviews, a separate section is dedicated to adopted inclusion criteria were: (1) studies published 2DMs beyond graphene: each material is briefly in English; (2) full-text articles; (3) cancer as target introduced and theranostic works discussed taking disease; (4) the presence of at least one type of GRM or into account of the design strategy, the type of cancer new emerging 2DMs in the considered nanosystem investigated, the working biological mechanisms structure; (5) studies with at least one diagnostic (when reported), the model used, the techniques, and method and one therapeutic strategy; (6) at least one the resulting outcomes. We include also emerging of these strategies was due to the presence of GRMs or 2DMs that did not prove their potentialities as new emerging 2DMs; (7) in vitro or in vivo studies in theranostic tools yet, but have promising appropriate animal models. The study selection characteristics for their future development in this required an initial stage in which the articles were direction. Moreover, the present review provides an selected, according to the eligibility criteria, based on overview and a critical discussion on the their title, abstract, and keywords. In the second stage, characterization performed for each study reported. the authors considered the full text of all the eligible In addition, tables and graphics were generated to studies, stating whether these met the eligibility indicate the types of cancer investigated in relation to criteria. Some of the first studies found in the the different approaches, the nanodrugs, and the literature were claiming possible theranostic forms of imaging. Furthermore, schematic views to applications, but only one application was compare the materials are also specified. The experimentally demonstrated; these studies were information collected in this review will allow the excluded by the present review. According to these readers to navigate among the 2DMs proposed for use criteria, the selected works for discussion on in cancer theranostics, discriminate which material is graphene, GRMs, and graphene hybrids for imaging more suitable for a specific theranostic aim, and combined with gene/drug delivery, PTT/PDT, or understand the oncological gaps and the remaining gene/drug delivery in association with PTT/PDT are open questions in 2D cancer theranostics. reported in Table 1, Table 2 and Table 3, respectively; Table 4 reports the published studies for the new 2DMs.

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Table 1. Characterization of all the studies using GRMs in cancer theranostics for combined imaging and drug delivery, on the basis of type of imaging, therapy, cancer, cell line, model, drug, gene, targeting moieties & other molecules, material and name of the nanotools.

GRAPHENE-RELATED MATERIALS Application Target Functionalization/coating Graphene-related material Reference Imaging Therapy Cancer Cell line Model Drug Gene Targeting Material Name moieties & other molecules Imaging MRI, CLSM Drug Breast cancer 4T1 In vitro, in DOX Suitable SPION GO CAD-SPIONs@GO Luo Y. et al. Chem Comm and drug/ delivery vivo for gene (2019) gene delivery delivery CLSM Drug Breast cancer MDA-MB In vitro DOX, - - rGO FA-rGO/ZnS:Mn QDs Diaz-Diestra D. et al. delivery 231 FA Nanomaterials (2018) MRI Drug Liver cancer HepG2 In vitro CA - Gd, Au GO BIT Usman M.S. et al. PLoS ONE delivery (2018) MRI Drug Liver cancer HepG2 In vitro PA - Gd, Au GO GAGPAu (or GOTs) Usman M.S. et al. Molecules delivery (2018)

CLSM Drug Breast cancer BT-474, In vitro DOX - HER and beta- GQDs GQD-NH2, GQD–βCD, Ko N.R. et al. RSC delivery MCF-7 cyclodextrin and GQD-comp, Adv.(2017) DL-GQD

MRI, CLSM Drug Cervical HeLa In vitro DOX - - GQDs Fe3O4@SiO2@GQD-FA/ Su X. et al Biosensors and delivery cancer DOX Bioelectronics (2017) CLSM, Drug Breast cancer 4T1 In vitro, in DOX - Cy GQDs DOX@GQD-P-Cy Ding H. et al. ACS Appl fluorescence delivery vivo Mater Interfaces (2017) imaging Fluorescence Drug Breast cancer MDA-MB- In vitro Apt - - GO MUC1 aptamer-NAS-24 Bahreyni A. et al. Int J Pharm microscopy, delivery 231, MCF-7 aptamer-GO, MUC1 (2017) flow cytometry aptamer-Cytochrome C aptamer-GO

MRI Drug Renal cancer 786-0 In vitro, in Apt and - Gd2O3, BSA GO GO/BSA-Gd2O3/AS1411 Li J. et al. J Biomed delivery vivo DOX -DOX Nanotechnol. (2016) Fluorescence Drug Bone cancer MG-63 In vitro, in PTX - ICG NGO NGO-PEG–ICG/PTX Zhang C. et al. RSC Adv. imaging delivery vivo (2016) CLSM, flow Drug Breast, cervical HeLa and In vitro BHC - - GQDs GQDs@Cys-BHC Thakur M. et al. Mater Sci cytometry delivery cancer MDA-MB- Eng C Mater Biol Appl. 231 (2016) CLSM Drug Lung, cervical, A549, In vitro Apt - - GQDs AS1411–GQDs Wang X. et al. J. Mater. delivery breast, liver HeLa, Chem. B (2015) cancer MCF-7, HepG-2 Intracellular Gene Cervical HeLa In vitro - - GQDs f-GQDs Dong H. et al ACS Appl microRNA deliery cancer miRNAs-2 Mater Interfaces. (2015) imaging 1 MRI, CLSM Drug and Brain cancer U87 In vitro, in EPI Let-7g - NGO Gd-NGO/Let-7g/EPI Yang H.W. et al. gene vivo, ex miRNA. Biomaterials. (2014) delivery vivo Optical Drug Liver cancer Bel-7402, In vitro DOX - - GO GO-RGD-Chitosan Wang C. et al Colloids and imaging delivery SMMC-772 Surfaces B: Biointerfaces 1, HepG2 (2014) Fluorescence Drug Lung cancer H1975 In vivo DOX - Cy5.5 GO GO-Cy5.5-Dox Nie L. et al. ACS Nano (2014) imaging PAI delivery Fluorescence Drug Colon cancer HCT116 In vitro, in Cur - - GQDs GQDs-Cur Some S. et al. Scientific microscopy delivery vivo Reports (2014) CLSM Drug Cervical HeLa and In vitro DOX - Lysotracker GO DOX@MSP-BA-GOF He D. et al Langmuir (2014) delivery cancer L02 Green Fluorescence Drug Cervical HeLa In vitro DOX - - graphene DOX-graphene-HQDs- Chen M.L. et al. imaging delivery cancer -HQDs Trf Bioconjugate Chem. (2013) CLSM, Raman Drug Cervical HeLa In vitro DOX - Au GO Au@NGO Ma X. et al. J. Mater. Chem. imaging delivery cancer B, 2013

MRI, CT Drug Cancer - Not tested - - Au/Fe3O4 rGO rGO/Au/Fe3O, and Chen Y. et al. ACS Nano. imaging delivery in and rGO/BaTiO3/Fe3O4 (2013) biological BaTiO3/Fe3O4 models MRI Drug Liver cancer HepG2 In vitro DOX - Gd(III) GO GO-DTPA-Gd Zhang M. et al. ACS Appl. delivery Mater. Interfaces (2013) MRI Drug Brain cancer U251 In vitro DOX - MGMSPID GO MGMSPI Wang Y. et al Small (2013) delivery Fluorescence Drug and Lung, prostate A549, In vitro, in DOX pDNA - GO DOX–CMG–GFP–DNA Wang C. et al. J Mater Chem imaging gene cancer LLC1, PC3, vivo B Mater Biol Med. (2013) delivery C42b PET Drug Breast cancer MCF-7 In vitro, in TRC105 - 64Cu rGO 64Cu-NOTA-rGO- Shi S. et al. Biomaterials delivery vivo, ex TRC105 (2013) vivo

MRI, Drug Liver cancer HepG2 In vitro DOX - SiO2 GO GO-SiO2 Gao Y. et al. Colloids and fluorescence delivery Surf. B Biointerfaces (2013) imaging PET Drug Breast cancer MCF-7, In vitro, in TRC105 - 64Cu GO 64Cu-NOTA-GO-TRC105 Hong H. et al. ACS Nano delivery 4T1 vivo (2012) Fluorescence Gene Cervical, HeLa and In vitro - pDNA BPEI GO GO-BPEI Kim H. et al. Bioconjugate imaging delivery prostate PC-3 (pCMV- Chem. (2011) Cancer Luc) Fluorescence Drug Burkitt's Raji B-cell In vitro DOX, - - NGO NGO-PEG/DOX + Sun X. et al. Nano Res. (2008) imaging, NIR delivery Lymphoma Rituxan Rituxan imaging

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Table 2. Characterization of all the studies using GRMs in theranostics for combined imaging, PTT and PDT, on the basis of type of imaging, therapy, cancer, cell line, model, PS, targeting moieties, material and name of the nanotools.

GRAPHENE-RELATED MATERIALS Application Target Functionalization/ Graphene-related material Reference coating Imaging Therapy Cancer Cell line Model PS Targeting Material Name moieties

Imaging MRI, CLSM, UCL, PTT, Cervical cancer Hela, U14 In vitro, - - GO GO/ZnFe2O4/UCNPs Bi H. et al. ACS publications and CT, PAT, UCLM PDT in vivo (GZUC) (2018) PDT/PTT UCL imaging PTT, Cervicalliver HeLa, U14 In vitro, - Ce6 NGO NGO-UCNP-Ce6 (NUC) Gulzar A. et al. Dalton Trans PDT cancer in vivo, ex (2018) vivo

X-ray CT imaging, PTT Cervical cancer HeLa In vitro, - - GO GO/Bi2Se3/PVP Zhang Y. et al. J. Mater. PAI in vivo Chem. B. (2017) NIR imaging, PAT PTT Skin cancer SCC7 In vitro, Au Cy5.5 GO CPGA Gao S. et al Biomaterials in vivo (2016) Fluorescence PTT, Skin cancer B16F0 In vivo - - NGO GO-PEG-folate-mediated Kalluru P. et al. Biomaterials imaging PDT NmPDT (2016) PAI PTT Brain cancer U87MG In vitro, - - rGO PEG-rGO-GSPs Lin L.S. et al. Nanoscale. in vivo (2016) CLSM, NIR PTT, Lung cancer A549 and Lewis In vitro, - - NGO NGO-808 Luo S. et al. ACS Appl Mater fluorescence and PDT lung cancer cells in vivo Interfaces. (2016) thermal imaging PAI PTT Breast cancer 4T1 In vitro, - ICG GO ICG-PDA-rGO Hu D. et al Theranostics. in vivo (2016) MRI, fluorescence PTT Sarcoma S180 In vitro, IO - GO IO/GO-COOH Huang G. et al Nanoscale imaging, IR in vivo (2015) thomography Raman PTT, Cervical cancer HeLa In vitro - GO PEG-Au@GON Kim Y.K. et al. Small. (2015) bioimaging PDT PAI PTT Breast cancer 4T1 In vitro, - - GO GO-PEG-CysCOOH Rong P. et al. RSC Adv. in vivo (2015) Fluorescence PTT, Lung cancer PC9 In vitro, - DVDMS GO GO-PEG-DVDMS Yan X. et al. Nanoscale. imaging, PAI PDT in vivo (2015) MRI, CT imaging PTT Cervical cancer HeLa In vitro, Gd(III) - GO GO/BaGdF5/PEG Zhang H. et al. Biomaterials. in vivo (2015) Fluorescence PDT Cervical HeLa, MDA In vitro - - GQDs NGs-QDs Ge J. et al Nature imaging Cancer MB-231 Communications (2014)

MRI, fluorescence PTT, Cervical HeLa In vitro SiNc4 - MGF MFG Gollavelli G. et al imaging PDT Cancer Biomaterials (2014)

Fluorescence PTT, Skin cancer G361 In vitro - ICG GO ICG-FeCl3 @GO Viraka Nellore et al. Faraday imaging PDT Discuss. (2014) Raman imaging PTT Breast Cancer SKBR-3 In vitro - - GO GO and GOAuNS Nergiz S.Z. et al ACS Appl. Mater. Interfaces (2014) CLSM, flow PTT, Breast cancer MDA-MB231 In vitro, - - cGdots cGdots Nurunnabi M. et al. ACS citometry, PDT in vivo Appl. Mater. Interfaces molecular imaging (2014) MRI PTT Pancreatic BxPC-3 In vitro, ION - GO GO-ION-PEG Wang S. et al Biomaterials cancer in vivo (2014) Fluorescence PTT, Breast cancer 4T1 In vitro, - HPPH GO GO-PEG-HPPH Rong P. et al. ADV imaging, PET PDT in vivo, ex Theranostics (2014) vivo NIR fluorescence PTT, Lung cancer A549 In vitro - Ce6 GO GO–HA–Ce6 Cho Y. et al. Chem Commun imaging PDT Camb (2013) MRI, CLSM PTT, Cervical cancer KB, HeLa In vitro, - - NGO UCNPs-NGO/ZnPc Wang Y. et al. Biomaterials. PDT in vivo (2013) MRI, X-ray CT PTT Cervical, KB, 4T1 In vitro, IONP-Au - GO GO-IONP-Au-PEG Shi X. et al. Biomaterials. breast cancer in vivo (2013) PAI PTT Cervical cancer Hela In vitro - ICG GO ICG-GO-FA Wang Y.W. et al. Journal of material chemistry B (2013) PAI PTT Breast cancer MCF-7 In vitro, - - NrGO NrGO Sheng Z. et al Biomaterials in vivo (2013) MRI, fluorescence PTT Breast cancer 4T1 In vivo IONP - rGO rGO–IONP–PEG Yang K. et al Advanced imaging, PAI Materials (2012) Fluorescence PTT Breast, cervical HeLa, MCF-7 In vitro, - - rGO rGO-QD Hu S.H. et al Advanced imaging cancer in vivo Materials (2012) Fluorescence PTT Breast cancer 4T1 In vivo - - NGS NGS-PEG Yang K. et al Nanoletters imaging (2010)

photothermal imaging. The multiple functions of 3.1. Graphene-related materials and hybrid these materials arise from their intrinsic nanosystems for cancer theranostics physicochemical properties and the possibility of The introduction of graphene and GRMs in the conjugating them with a wide variety of molecules field of theranostics has allowed the combination of [48]. We here discuss the different approaches used, effective therapeutic procedures (e.g., relying on starting from less complex nanotools, with only two targeted drug/gene delivery, PTT, PDT, etc.) with a applications (e.g., imaging and gene/drug delivery), wide range of different imaging methods, such as to more and more complex theranostic tools with MRI, PET, CT, fluorescent imaging, PAI, and several multiple functions, where imaging,

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5441 gene/drug delivery, and PTT/PDT are combined in a subsequent explosion of graphene-related research for unique nanotheranostic platform. All the works taken cancer theranostics. Several studies further involved into consideration are reported in Table 1 (imaging the chemical functionalization of graphene, GRMs, and drug/gene delivery), Table 2 (imaging and and graphene hybrids to improve and expand their PTT/PDT) and Table 3 (imaging and drug/gene use for gene/drug delivery and imaging [49–77]. In delivery in association with PTT/PDT). particular, DOX represented the first-choice chemotherapeutic agent also for other authors. For 3.1.1. Imaging and drug/gene delivery example, GO was functionalized by Ten years ago, Sun et al. introduced the use of a magnetic/fluorescent SiO2 microsphere through an GRM in cancer theranostics, exploring a single-layer amidation process and loaded with DOX, creating an NGO-based platform for combined diagnosis and active fluorescent magnetic drug carrier and a therapy [49]. The material, showing intrinsic potential optical imaging tool [75]. Similarly, Ma and photoluminescence (PL) exploitable for live cell co-authors successfully used Au-decorated GO NPs imaging in the NIR, was coated by polyethylene for combined DOX delivery and intracellular Raman glycol (PEG) to improve its solubility and imaging for cervical cancer (HeLa cells) [69,78]. biocompatibility. Moreover, NGO imaging properties Thanks to the quenching of DOX fluorescence were combined with its loading capability, resulting induced by the attachment to the GO-nanosystem, it in a theranostic nanoplatform. To this end, the was possible to track the delivery of the drug that was anticancer drug DOX was bound to NGO sheets, able to emit only when released in the tumor cells. which were functionalized with the B cell specific Theranostic GO-based DOX nanocarriers were also antibody Rituxan (anti-CD20) for the in vitro selective fabricated by Nie et al. for combined drug delivery binding and killing of Burkitt’s lymphoma cells. A and PAI in lung cancer cells (H1975 cells) and in vivo. sustained Raji B cell growth inhibition ( 80%) was The system was also linked to the Cy5.5 dye to allow observed after 2-h incubation with the nanosystem at fluorescence imaging. Moreover, thanks to the high ∼ DOX concentration of 10 µmol/L followed by 48-h loading capacity, the material was able to induce incubation in fresh cell medium. effective PAI-monitored chemotherapy in mice [65]. This promising work paved the way for the

Table 3. Characterization of all the studies using GRMs in theranostics for combined imaging, drug delivery, PTT and PDT, on the basis of type of imaging, therapy, cancer, cell line, model, drug, gene, PS, targeting moieties, material and name of the nanotools.

GRAPHENE-RELATED MATERIALS Application Target Functionalization/coating Graphene-related material Reference Imaging Therapy Cancer Cell line Model Drug Gene PS Targeting Material Name moieties Imaging, MRI Drug Breast 4T1 In vitro MTX - Mn(II) DTPA rGO rGO-PDA-BSA-DTPA Karimi Shervedani R. et drug delivery, PTT cancer Mn(II)/MTX al. Biosens Bioelectron. delivery (2018) and MRI, PAI Drug Breast 4T1 In DOX - MnWO4 - GO GO/MnWO4/PEG Chang X. et al. Carbon PTT/PDT delivery. PTT cancer vitro, (2018) in vivo CLSM, UCL Drug Cervical, Hela, U14 In DOX - - FITC NGO UCNPs-DPA-NGO-PEG- Gulzar A. et al. Dalton imaging delivery, PTT liver vitro, BPEI-DOX Trans (2018) cancer in vivo CLSM Gene Lung, A549, In vitro - miRNA - - GQDs GQD-PEG-P Cao Y. et al. ACS Appl. delivery, PTT, breast MCF-7 Mater. Interfaces (2017) PDT cancer CLSM, Drug Breast EMT6 In - - - Ce6 GO GO/AuNS-PEG and Wu C. et al. Acta thermal/PT delivery, PTT, cancer vitro, GO/AuNS-PEG/Ce6 Biomater. (2017) imaging PDT, in vivo sonodynamic therapy CLSM, NIR Drug delivery Breast B16F10, In - - - PheoA GO PheoA + GO:FA-BSA-c- Battogtokh G. et al. fluorescence PTT, PTT cancer MCF-7 vitro, PheoA NC Journal of Controlled imaging in vivo Release (2016) CLSM Drug Cervical HeLa In vitro LH - - - rGO, MGQDs-LH Justin R. et al. Carbon delivery, PTT cancer GQDs (2016) Fluorescence Drug Lung A549 In vitro anti‐ - - - rGO anti‐EGFR‐PEG‐rGO@CPSS‐ Chen Y.W. et al. Small imaging, delivery, PTT cancer EGFR Au‐R6G (2016) CLSM, SERS SERS imaging, probes Optical imaging, Raman imaging NIR imaging, Drug Breast 4T1 In DOX - - PANI GO GO-Au@PANI/DOX Chen H. et al. SERS delivery, PTT cancer vitro, Theranostics (2016) in vivo MRI Drug Breast MCF-7 In DOX - Gd(III) - GO GO@Gd-PEG-FA/DOX Shi J. et al. Pharm Res. delivery, PTT cancer vitro, (2016) in vivo

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GRAPHENE-RELATED MATERIALS Application Target Functionalization/coating Graphene-related material Reference Imaging Therapy Cancer Cell line Model Drug Gene PS Targeting Material Name moieties Molecular Drug Cervical KB In - - - ICG rGO ICG/HArGO and ICG/rGO Miao W. et al. J Control imaging, delivery, PTT cancer vitro, Release. (2015) real-time IR in vivo thermal imaging Fluorescence Drug Ovarian A2780/AD In - - - Pc LOGr LOGr-Pc-LHRH Taratula O. et al. Int. J. imaging delivery, PTT, cancer vitro, Nanomed (2015) PDT in vivo Fluorescence Drug Brain U87MG In - - - DVDMS GO GO-PEG-DVDMS Yan X. et al. Biomaterials. imaging delivery, PDT cancer vitro, (2015) in vivo, ex vivo Fluorescence Drug Cervical Hela In vitro DOX - - - rGO DOX–rGO–Fe2O3@Au NPs Chen H. et al. RCS Adv. imaging, MRI delivery, PTT cancer (2015)

Fluorescence Drug Cervical HeLa In vitro HA - - GQDs HA–GQD–SiO2 Zhou L. et al. Chem. imaging delivery, PDT cancer Commun. (2015) Photothermal Drug Lung A549 In DOX - - - Graphene GDH Khatun Z. et al. imaging, delivery, PTT cancer vitro, Nanoscale (2015) optical in vivo imaging MRI Drug Cervical HeLa In DOX - MnFe2O4 - GO GO/MnFe2O4/DOX Yang Y. et al. Journal of delivery, PTT cancer vitro, Biomaterials in vivo, Applications (2015) ex vivo TPL Drug Breast MCF-7 In vitro DOX - Au - graphene NGsAu nanocrystal Bian X. et al Scientific delivery, PTT cancer Reports (2014) CLSM Gene Breast MCF-7 In vitro DOX - - FITC NGO GO-PEG-DA Feng L. et al Adv Healthc delivery, PTT cancer and DAPI Mater. (2014) CLSM Drug delivery Breast MCF-7 In DOX - Ag - GO GO-Ag Shi J. et al Biomaterials PTT cancer vitro, (2014) in vivo Fluorescence Drug Cervical Hela In PLA - - - GO Au@PLA-(PAH/GO)n Jin Y. et al. Biomaterials imaging, X-ray delivery, PTT cancer vitro, (2013) CT imaging, in vivo US imaging CLSM Drug Brain U251 In vitro DOX - - - Graphene GSPI Wang Y. et al delivery, PTT cancer J.Am.Chem.Soc. (2013) UCL imaging Drug Breast KB, HeLa In ZnPc - - ZnPc GO GO-UCNPs--ZnPc Wang Y. et al delivery, PTT, cancer vitro, Biomaterials (2013) PDT in vivo, ex vivo Molecular Drug Skin SCC7 In DOX - - Ce6 GO Ce6/Dox/pGO Miao W. et al imaging delivery, PTT cancer vitro, Biomaterials (2013) in vivo

Table 4. Characterization of all the studies using 2DMs in cancer theranostics, on the basis of type of imaging, therapy, cancer type, cell line, model, drug, PS, targeting moieties, material, and name of the 2D tools.

NEW 2D MATERIALS Applications Target Functionalization/coating 2D materials Reference Imaging Therapy Cancer Cell line Model Drug Other molecules/ particles

WS2 PAI, MRI, PTT and Breast cancer 4T1 In vitro, - IONPs, MnO2 WS2-IO/S@MO-PEG Yang G. et al. fluorescence radiotherapy in vivo Small (2018) imaging 188 188 SPECT, IR PTT and Breast cancer 4T1 In vitro, - Re Re-WS2-PEG Chao Y. et al. thermal and radiotherapy in vivo Small (Weinheim an der fluorescent Bergstrasse, Germany, 2016) imaging

CT, IR and PTT, PDT Cervical cancer HeLa In vitro, - Bovine serum albumin BSA-WS2@MB Yong Y. et al. fluorescence in vivo (BSA), Nanoscale (2014) imaging methylene blue

PAT, CT, IR PTT Cervical and 4T1, HeLa and In vitro, - LA-PEG WS2-PEG Cheng L. et al. imaging breast cancer 293T in vivo Advanced Materials (2014)

MoS2 MRI, CLSM, flow Drug delivery, PTT Lung cancer A549, H1975 In vitro, Gefitinib Hyaluronic acid (HA), MoS2-HA-DTPA-Gd Liu J. et al. cytometry in vivo gadolinium (Gd), DTPA Journal of Colloid and Interface Science (2019)

NIR fluorescence Drug delivery, PTT Liver cancer LO2, Hep3B In vitro MET Mn-doped Fe3O4, Mn-doped Jing X. et al. Bioconjugate imaging chitosan Fe3O4@MoS2@CS Chemistry (2018)

Two-photon PTT, PDT Cervical cancer HeLa In vitro - AuNBPs AuNBPs@MoS2 Maji S. et al. ACS Applied CLSM and Materials and Interfaces fluorescence (2018) imaging

IR, PET, FLIM, Drug delivery, PTT Breast and lung A549, MCF-7, In vitro, DOX PEI, HA MoS2-PEI-HA Dong X. et al. ACS Applied Flow cytometry cancer MCF-7-ADR in vivo Materials and Interfaces (2018)

MRI, PAI, CLSM PTT Breast cancer 4T1, RAW In vitro, - Bovine serum MoS2-Gd-BSA Chen L. et al. 264.7, L929 in vivo albumin-gadolinium ACS Applied Materials and (BSA-Gd) Interfaces (2017) MR, IR, and PA PTT, PDT and Hepatoma and L929 In vitro, MoS2@Fe3O4-ICG/Pt(I Liu B. et al. Advanced imaging chemotherapy cervical cancer in vivo V) Nanoflowers Science (2017)

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NEW 2D MATERIALS Applications Target Functionalization/coating 2D materials Reference Imaging Therapy Cancer Cell line Model Drug Other molecules/ particles MRI, CT, CLSM, PTT, PDT Cervical cancer HeLa, L929 In vitro, - chlorin e6 (Ce6), UCS MUCS–FA Xu J. et al. UCLM in vivo Small (2017)

PAI, NIR PTT Colorectal cancer HT29, L929 In vitro, - PVP MoS2-PVP Zhao J. et al. Oncotarget in vivo (2017)

IR and phase PTT Breast cancer 4T1, L929 In vitro, - Soybean phospholipid SP-MoS2 Li X. et al. International contrast imaging in vivo Journal of Nanomedicine (2016)

MRI, PAT and PTT Cervical and liver HeLa, HepG2 In vitro - Fe3O4, PEG MSIOs Yu J. et al. Theranostics fluorescence cancer (2015) imaging

PAI, CT, IR PTT and Breast cancer 4T1 In vivo - Bi2S3 MoS2/Bi2S3 Wang S. et al. radiotherapy Advanced Materials (2015) 64 64 PAT, MRI, IR, PTT Breast cancer 4T1, RAW 264.7 In vitro, - LA-PEG, IONPs, Cu Cu-MoS2-IO-(d)PEG Liu T. et al. PET in vivo ACS Nano (2015)

CLSM, IR, flow PTT, PDT Breast cancer 4T1 In vitro, - Ce6, LA-PEG MoS2-PEG Liu T. et al. cytometry in vivo Nanoscale (2014) BP PAI, fluorescence PTT Breast and lung A549, MCF-7, In vitro, - RGD RP-p-BPNSs Li Z. et al. ACS Applied imaging cancer LO2 in vivo Materials and Interfaces (2019)

MR, IR, CLSM PTT, PDT Cervical cancer HeLa In vitro, - Fe3O4-CDs, GP, PGA GP-PGA-Fe3O4-CDs@BP Zhang M. et al. International in vivo QDs Journal of Nanomedicine (2018)

MRI, PDT and Melanoma A375 In vitro, - Bi2O3 BP/Bi2O3 Huang H. et al. Biomaterials fluorescence radiotherapy in vivo (2018) imaging, flow cytometry IR thermal, PTT Cervical cancer HeLa In vitro, - - BPQDs Wang M. et al. Analyst CLSM in vivo (2018)

MRI, IR, CLSM Drug delivery, PTT Breast and lung A549, MCF-7 In vitro, DOX Fe3O4@C, SiO2 BPQDs@ss-Fe3O4@C Zhang M. et al. Chemistry - cancer in vivo A European Journal (2018) IR thermal, Drug delivery Breast cancer MDA-MB-231 In vitro, DOX - BP@Hydrogel Qiu M. et al. Proceedings of fluorescence in vivo the National Academy of imaging Sciences (2018) IR thermal, PTT, PDT Cervical cancer HeLa In vitro, - PEG, Ce6 BP@PEG/Ce6 NSs Yang X. et al. ACS Applied fluorescence in vivo Materials and Interfaces imaging (2018) IR thermal, PTT Osteogenic Saos-2 In vitro, - Bioglass (BG) BP-BG scaffold Yang B. et al. Advanced CLSM sarcoma in vivo Materials (2018) IR thermal, Drug delivery, Breast cancer 4T1 In vitro, DOX - BP Chen W. et al. Advanced fluorescence PTT, PDT in vivo Materials (2017) imaging

PAI PTT, PDT Breast cancer MCF-7 In vitro, - TiL4 TiL4@BPQDs Sun Z. et al. Small (2017) in vivo NIR, CLSM Drug delivery, PTT Cervical cancer HeLa In vitro, DOX PEG, Cy7 BP-PEG-FA/Cy7 NSs Tao W. et al. Advanced in vivo Materials (2017) IR thermal, PTT, PDT Liver and breast HepG2, 4T1 In vitro, - RdB, PEG RdB/PEG-BPQDs Li Y. et al. ACS Applied CLSM cancer in vivo Materials and Interfaces (2017) IR thermal PTT Breast cancer 4T1 In vitro, - Au BP-Au NSs Yang G. et al. Biomaterials imaging in vivo Science (2017) PAI, IR thermal, PTT Breast cancer 4T1 In vitro, - PEG PEGylated BP Sun C. et al. Biomaterials and fluorescence in vivo (2016) imaging

MXene IR thermal, PAI, PTT and photonic Breast cancer 4T1 In vitro, - AIPH, SiO2 AIPH@Nb2C@mSiO2 Xiang H. et al. CLSM, thermodynamic in vivo ACS Nano (2019) fluorescence therapy imaging, flow cytometry

PAI, CT, CLSM, PTT and Breast cancer 4T1 In vitro, - Au Ti3C2@Au Tang W. et al. IR thermal radiotherapy in vivo ACS Nano (2019)

MRI, CT, CLSM PTT Breast cancer 4T1 In vitro, - Soybean phospholipid, Ta4C3-IONP-SPs Liu Z. et al. in vivo IONP Theranostics (2018) PAI, CLSM, IR, PTT Brain cancer U87 In vitro, - CTAC, RGD CTAC@Nb2C-MSN Han X. et al. flow cytometry in vivo Theranostics (2018) PAI, IR, CLSM Drug delivery, PTT Liver cancer HCC, In vitro, DOX RGD Ti3C2@mMSNs Li Z. et al. SMMC-7721 in vivo Advanced Materials (2018)

PAI, fluorescence Drug delivery, PTT Breast cancer 4T1 In vitro, DOX Soybean phospholipid Ti3C2-SP Han X. et al. imaging in vivo Advanced Healthcare Materials (2018)

IR thermal, PAI PTT Breast and brain 4T1, U87 In vitro, - PVP Nb2C-PVP Lin H. et al. and fluorescence cancer in vivo Journal of the American imaging Chemical Society (2017)

IR thermal, PAI PTT Cervical cancer HeLa In vitro - - Ti3C2 QDs Yu X. et al. and in Nanoscale (2017) vivo

MRI, CT, PAI, PTT Breast cancer 4T1 In vitro - MnOx, soybean MnOx/Ta4C3-SP Dai C. et al. CLSM and in phospholipid ACS Nano (2017) vivo

CLSM, IR PTT, PDT Colon cancer HCT-116 In vitro DOX HA Ti3C2-DOX Liu et al. AM&I (2017) thermal, and in fluorescence vivo

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Figure 1. Graphical representation of the theranostic applications of different GRMs and 2DMs beyond graphene.

Different studies have succeeded in exploiting suppression in mice. GQDs fluorescence for targeted cancer imaging and, Moreover, graphene has attracted increasing at the same time, for tracking and monitoring drug attention in MRI-based theranostic protocols. As a delivery processes and cancer therapies. For example, non-invasive diagnostic technology, MRI has been GQDs have proven to be an optimal multifunctional widely used in the clinic; however, challenges nanovehicle for delivering DOX to targeted cancer associated to biocompatibility and sensitivity of the cells, enabling the monitoring of the intracellular contrast agents used in MRI and in anticancer drug release as a dual-fluorescent nanotechnology-based approaches still need to be nanoprobe [54,56,60–62,66,68,79]. It has been solved [80]. In 2013, Zhang et al. developed a positive underlined how the drug-loaded stereochemistry can T1 MRI GO-contrast agent GO−DTPA−Gd/DOX affect GQD imaging behavior [66]. In this case, based on GO-gadolinium (Gd) complexes. This ⦋ ⦌ anticancer drug curcumin (Cur) chelated the nanocomplex offers a dual-modality: T1 fluorescence of GQDs until the release into the tumor MRI/fluorescence imaging and drug delivery site, allowing restoring of the fluorescence of GQDs, functionalities, which exhibited low cytotoxicity. The hence acting as a bio-probe for tumor imaging. GQDs developed MRI contrast agent can be internalized into were also explored as efficient nucleic acid cells, enabling cellular MR imaging. nanocarriers for the regulation of intracellular Moreover, GO−DTPA−Gd allows a high miRNAs and imaging [62]. In this case, the multiple capacity DOX loading, resulting in potent anti-cancer gene probes loaded on GQDs, showed a combined activity against HepG2 cells [71]. Li et al. fabricated a effect for the enhancement of the therapeutic efficacy. GO/BSA-Gd2O3/AS1411-DOX theranostic nanocom- The uptake of the GQDs by HeLa cells was monitored plex with BSA-Gd2O3 NPs intended to be used as an by exploiting the intrinsic PL of GQDs, while the MRI contrast agent, where graphene oxide nanoplates fluorescence of the gene probe, produced by the (GONs) are used as both contrast agents and drug recognition of the target, was used to monitor the nanocarrier, conjugated with an aptamer, AS1411, regulation of the target gene. which serves as the targeting molecule [57]. This Paclitaxel (PTX) was also explored for drug theranostic nanocomplex has shown not only an delivery by Zang et al., which used indocyanine green increased MR contrast signal, but also a specific (ICG)-loaded NGO for combined PTX shuttle at the targeting and growth inhibition of human renal tumor site and fluorescence imaging [58]. In vivo data carcinoma 786-0 cells, demonstrating drug delivery demonstrated that the system was highly ability both in vitro and in vivo. Working in a similar biocompatible and able to induce a total cancer direction, in 2018, Usman et al. developed a bimodal

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5445 theranostic nanodelivery system (BIT) suitable for µg/mL of DOX, far below the values frequently combined and simultaneous MRI and drug delivery reported in the literature (>10 µg/mL). Chen et al. [52]. This nanoplatforms consisted of GO, chlorogenic developed a rGO-based nanoplatform based on acid as a chemotherapeutic agent, gadolinium (Gd), rGO/Au/Fe3O4 hybrids, used as cargo-filled and gold nanoparticles (AuNPs) as contrast agents for graphene nanosacks that when reintroduced into the MRI. The authors reported targeting and growth aqueous environment can rapidly release soluble salt inhibition of hepatocellular carcinoma HepG2 cells, cargoes. These open structures can be adaptable to a and the nanoplatform was shown to produce a drug-controlled release form by adding a polymeric stronger signal than the conventional MRI contrast filler. To apply the theranostic use on this system, the agents (Gd(NO3)3). The obtained results portray this authors combined a magnetically responsive system as a promising future chemotherapeutic for platform, deonstrating an optimal contrast cancer treatment with MRI diagnostic modalities [52]. enhancement as imaging probes in both MR imaging The same group also developed another theranostic and X-ray computed tomography [70]. system for MRI, the so-called GOTS system, which One of the most promising strategies for the consisted of GO, protocatechuic acid as an anticancer treatment of cancer consists of combining agent, Gd (III) nitrate hexahydrate combined with chemotherapy with gene therapy. In this view, in the AuNPs as a diagnostic agent. The system was able to effort of creating a single platform, able to efficiently induce cancer cell HepG2 death at a concentration of deliver genes, drugs and contrast agents to the cancer 100 µg/mL. The T1 weighted image of GOTS showed site, Wang et al. reported a functionalized chitosan increment in contrast to the nanocomposite to be magnetic graphene (CMG) system for the higher than pure (Gd(NO3)3 and water reference. simultaneous delivery of gene/drugs and SPIO y to These initial in vitro outcomes suggest an upcoming tumors. Ex vivo MRI demonstated the use of CMG as a solution to the highly toxic chemotherapy and strong T2 contrast-enhancing agent. As shown by diagnosis of cancer diseases [53]. In 2019, Yu Luo et al. biodistribution studies and MRI, CMGs selectively developed ultrasmall and superparamagnetic iron accumulated in tumors. The system has been reported oxide nanoparticles (IONPs), loaded on GO to show an efficient drug loading capacity, nanosheets [SPIONs@GO], for T1-MR imaging and pH-dependent drug/gene release and better pH-sensitive chemotherapy of tumors [50]. They cytotoxicity than free DOX. Moreover, DOX–CMG– showed a sensitive and modulable pH-responsive GFP–DNA NPs were able to deliver both DOX and drug release behavior triggered by even subtle pH GFP coding pDNA to the tumor site in mice, serving alterations. In vivo results further confirmed as an integrated system of targeted imaging, high-resolution T1-weighted MR imaging drug/gene co-delivery and real-time monitoring of performance and high antitumor efficacy [50]. therapeutic effects [73]. Functionalization with folic acid (FA) was introduced with the aim of both improving drug 3.1.2. Imaging and photothermal/photodynamic selectivity and reducing the material related toxicity therapy towards healthy cells. For examples, He et al. designed Compared to conventional treatment methods, a GO-capped mesoporous silica nanoplatform PTT and PDT exert a selective and non-invasive (MSP-BA-GO) for remote-controlled drug release, anticancer action. The former refers to the use of combined with DOX-loading and folic acid electromagnetic radiation, such as NIR wavelengths, modification [67]. DOX@MSP-BA-GOF displayed a to excite a PS. The absorption of a specific band light selective cellular internalization via receptor- leads the PS to an excited state. Coming back to the mediated endocytosis and the subsequent release of steady-state, it releases energy in the form of heat, DOX by remote illumination. In another study, leading to cancer cell photoablation. To gain this effect Diaz-Diestra et al. demonstrated that the FA and to avoid nonspecific killing of healthy cells, PSs functionalization of rGO-based nanoplatform need to absorb in the NIR and be selectively uptaken (FA-rGO/ZnS:Mn) improved targeting of the folate into cancerous cells [81]. On the other hand, in PDT, receptor-positive cancer cells and inhibited the PSs absorb light and transfer the energy to the oxygen toxicity exerted on non-tumor cells up to 72 h present in the surrounding tissue. The production of 1 exposure [51]. This was due to the surface passivation highly reactive oxygen species (ROS), such as O2 and of FA, which allowsto decrease the strong free radicals, oxidizes cellular and sub-cellular hydrophobic interaction between cell membrane wall structures, such as plasma, lysosomal, mitochondrial, and graphene flakes edges or corners, which are and nuclear membrane, leading to non-recoverable proven to induce toxic responses. The nanosystem damage to tumor cells [82]. killing efficiency was 50% at a concentration of 3 Currently, a large number of nanomaterials are

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5446 being studied for PTT and PDT applications, thanks to use for passive tumor targeting and PAI. Cancer cells, their optical absorbance in the NIR, including AuNPs in tumor-bearing mice, were efficiently ablated due to and GRMs [73,74,83–110]. From a theranostic point of the photothermal effect of NrGO after view, photothermal agents (PTAs) are very appealing continuous-wave NIR laser treatment [107]. since they can also serve as contrast agents for PAI, Other studies selected GO as starting material representing a noninvasive imaging modality thanks for their theranostic platform for imaging in to its high spatial resolution and outstanding association with PTT/PDT [73,74,84–96,99,100,102– soft-tissue contrast [111,112]. 109]. For example, Wang et al. decorated the material The creation of advanced graphene-based with ICG as the photoresponsive imaging agent and multi-modal nanosystems for combined diagnosis FA as a targeting moiety [114]. The resulting complex and therapy has paved the way for new theranostic (ICG–GO-FA) exhibited a high optical absorbance in protocols. In this perspective, graphene is very the NIR region, endowing it with excellent attractive as PS agent or as a carrier for PSs for a photothermal properties. In vitro data demonstrated photothermal and photodynamic alternative that 1 h incubation with ICG–GO–FA (20 µg/mL), approach in cancer therapy, due to its excellent followed by 808 nm NIR laser irradiation, could thermal properties and electrical conductivity [113]. induce a targeted photothermal HeLa cervical cancer In 2010, Yang et al. assessed the in vivo effects of cell death (8% residual cell viability) in association nanographene sheets (NGS) coated by PEG and with PAI. In another study exploring GO potential for labeled with a fluorescent method [110]. The cancer theranostics, the PAI signals as well as the GO fluorescence imaging showed a high NGS tumor NIR absorbance were dramatically enhanced by the uptake for several xenografted tumor mouse models. functionalization of the material with the NIR PEGylated NGS displayed an impressive in vivo fluorescence dye CySCOOH, showing a complete behavior, including increased tumor targeting PTT-induced tumor ablation in vivo without any sign efficiency and low reticuloendothelial retention. The of recurrence in the next 60 days of follow up [93]. conjugation with PEG further improved the Another GO-based theranostic platform for photothermal activity. The NGS strong optical combined PAI and PTT was developed by Gao et al. absorbance in the NIR region allowed their use for [80]. The authors studied a GO/gold-based probe PTT, achieving an optimal tumor ablation after their (CPGA) with enhanced NIR absorbance and intravenous administration and tumor irradiation photoconversion efficiency applicable in multimodal with low-power NIR laser. fluorescence imaging and photoacoustic The work of Li et al. represents a recent image-guided PTT of cancer. The intravenous contribution for cancer diagnosis in combination with administration of CPGA into tumor-bearing mice PTT, reporting the development of multifunctional resulted in the observation of tumor localized high NGO-based composite (UCNP@NGO), complexed fluorescence and PA signals. Moreover, laser with upconversion nanoparticle NaLuF4:Er3+, Yb3+, exposure caused tumors growth inhibition and with high photothermal conversion efficiency in ablation. One more successful procedure achieving association with UCL imaging [84]. Both in vitro and total cancer elimination in mice after 808 nm laser in vivo data demonstrated UCNP@NGO excellent irradiation was obtained by loading of Bi2Se3 NPs on biocompatibility and high theranostic effectiveness GO in the presence of polyvinylpyrrolidone (PVP) inhibiting tumor growth. [87]. The high biocompatible nanosystem served a as Also, nano-reduced graphene oxide (NrGO) was an outstanding bimodal imaging (CT and PAI) in explored for imaging in association with PTT. association with PTT platform for imaging-guided Compared to NGS and NGO, NrGO has a higher therapy, without any sign of tumor re-growth up to 24 photothermal activity due to the intrinsic days. Impressive system imaging ability in association physicochemical properties and uniform dispersivity. with phototherapy was also reported in the study of In fact, NGO reduction to NrGO causes an increased Bi et al., where the designed GO-based nanoplatform, degree of π conjugation, producing an amplified NIR supported by ZnFe2O4 and UCNPs, was able to absorption with enhanced photothermal activity. In perform a quad-model imaging-guided PTT/PDD, 2013, Sheng et al. developed a new protein-based exploiting its MRI, CT, UCL and PAT capability and method for the fabrication of NrGO, demonstrating its obtaining a sustained tumor reduction in mice [85]. ability as a highly integrated theranostic agent for Ray et al. explored GO for highly selective and photoacoustic (PAI)/ultrasonic (US) dual-modality ultra-sensitive melanoma cancer cell detection from imaging and PTT. Systematic administration of NrGO blood samples [99]. To this end, an AGE-aptamer- displayed an optimal photoacoustic signal conjugated magnetic hybrid GO-based assay was enhancement in the tumor area, paving its possible used as a multicolor luminescence system for tumor

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5447 cells and attached with ICG for combined PTT/PDT collaborators exploited the conjugation with PEG, using a 785 nm laser irradiation. In the presence of iron oxide and AuNPs developing GO-IONP- NIR light the system was highly effective, indicating Au-PEG, as a powerful photothermal agent for in vitro good performances of the material in inhibiting tumor cancer cell killing using molecular or magnetic growth, while any reduction of cell viability was targeting [116]. Thanks to the subsequent in vivo observed in the absence of the irradiation after 12 h studies, they were able to demonstrate the efficacy of exposure, demonstrating the biocompatibility of the this dual model imaging-guided photothermal tumor nanoplatform. A novel photo-theranostic platform destruction method, proving an excellent tumor based on sinoporphyrin sodium (DVDMS) loaded ablation. The study suggested that the IONP and Au PEGylated GO was studied in 2015 by Yan et al. The substituents in the GO-IONP-Au-PEG nanocomposite GO-PEG carrier improved the fluorescence of structure could be further exploited for MR and X-ray DVDMS through intramolecular charge transfer as dual-modal imaging. well as the tumor accumulation of DVDMS. The NIR The adoption of iron oxide could be used in absorption of GO was enhanced by DVDMS, leading order to increase in T2 contrast enhancement. For this to improved PAI and PTT. In vivo results showed that reason, many studies developed graphene-iron oxide systemic administration of the system could even NPs with improved imaging properties. Wang et al. result in total tumor eradication [94]. Kalluru et al. developed GO-iron oxide NPs, as a nanotheranostic reported single-photon excitation wavelength- agent for the diagnosis and treatment of regional dependent photoluminescence in the visible and short lymph node (RLN) metastasis of pancreatic cancer NIR region [88]. When authors analyzed the [102]. Intratumoral injection of GO-iron oxide NP 1 formation of O2, it was shown that the system is resulted in its transportation to RLN via lymphatic suitable for the in vivo fluorescence imaging operated vessels led to the regional lymphatic system using inexpensive laser setups using low laser doses. dual-modality mapping through MRI and to efficient By combining PEG and folate, nano-sized GO has tumor ablation. PEGylation of the system allows been shown to effectively result in PDT and PTT in achieving lower systematic toxicity, suggesting that vitro and in vivo using NIR light at ultra-low doses. these efficient theranostic nanoplatforms can be Yang and co-workers developed another promising engineered to be safer for future clinical studies. gold-based GO/BaGdF5/ PEG usable as a Huang et al. tested iron oxide/GO-COOH T1-weighted MR and X-ray CT dual-mode contrast nanocomposites with high photothermal conversion agent [96]. GO/BaGdF5/PEG demonstrated to be an efficiency and enhanced contrast [91]. The authors optimal photothermal agent for in vivo PTT cancer reported an effective inhibition of tumor growth due treatment due to its strong NIR absorbance and an to the improved photothermal effect. improved contrast agent providing MR/CT bimodal Only three studies were based on the use of imaging-guided therapy. Other studies further GQD-based nanoplatform for combined imaging and inspired the application of GO/gold hybrid PTT/PDT [83,97,101]. For example, a theranostic nanocomposites for image-guided enhanced PTT in probe based on SPIO and bismuth oxide (Bi2O3) with biomedical applications. In 2014, Nergiz et al. GQD coating was fabricated by the group of Mesbahi validated a novel class of multifunctional A for in vitro CT/MR dual-modal biomedical imaging graphene/gold hybrid nanopatches consisting of GO and guided PTT [83]. A high inhibitory effect on and gold nanostars (GO-AuNS) for an improved cancer cells proliferation was reported after the image-guided PTT [100], whereas Jin et al. (2013) co-treatment with GQDs-Fe/Bi NPs and NIR developed Au@PLA-(PAH/GO)n microcapsules as irradiation, demonstrating the exceptional multifunctional theranostic agent, acting as contrast performance of this theranostic nanoplatform for MR agent for both ultrasound (US) imaging and X-ray CT imaging, high-contrast CT imaging, and CT imaging and showing exceptional photoablation enhancement efficiency. effectiveness, as suggested by the photothermal The use of graphene and GRMs in PDT directed experiments [115]. The use of US/CT bimodal theranostics have also been reported in various imaging allowed to obtain an enhanced imaging studies, however to a lesser extent compared to PTT contrast and specific tumor anatomic information. involving applications. For what concerns the The contrast imaging was applied to identify the adoption of graphene in the PDT protocols, in 2014, location and size of the tumor, while NIR Ge et al. exploited the intrinsic GQDs properties, such laser-induced photothermal target therapy was as a broad absorption from the visible to the NIR, carried out based on the diagnostic imaging results, deep-red emission, high photo- and pH-stability and avoiding damaging healthy tissues. To further biocompatibility, for imaging purposes in association 1 improve the photothermal activity, Shi and with PDT [97]. GQDs exhibited a high O2 generation

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5448 yield, enabling them to be used as in vivo PTT and PDT and bioimaging. Results also suggested multifunctional graphene-based nanoplatform for that ZnPc-PEG-Au@GON NPs resulted in low simultaneous imaging and extremely efficient PDT of cytotoxicity [92]. Luo et al. combined the PS IR-808 different types of cancer, including skin melanoma with NGO and studied its PDT, PTT, and imaging and tumors located near the skin. capabilities [90]. Authors achieved high tumor In cancer treatment, researchers are frequently accumulation by targeting organic-anion transporting motivated to combine different modalities to achieve polypeptides (OATPs) overexpressed in many cancer an efficient cancer diagnosis and therapy. New cells. Results suggested that this system (NGO-8080) advances have been made to establish a targeted can provide high-performance cancer phototherapy protocol that covers the simultaneous application of with minimal side effects through the synergistic imaging methods and PTT or PDT. The theranostic PDT/PTT treatment and cancer-targeted progress made by the previously cited studies led to accumulation. In 2018, Gulzar et al. covalently the development of new combined protocols implanted upconversion NPs (UCNPs) with involving graphene or GRM-based nanoplatforms for PEGylated NGO and loaded the system with the PS simultaneous imaging, PTT, and PDT approach. Ce6 [86]. The authors reported a significantly In 2013, a promising integrated probe was enhanced and synchronized therapeutic effect developed for upconversion luminescence (UCL) paralleled to the individual PTT or PDT. Therefore, image-guided combinatorial PDT/PTT of cancer this study showed that this multifunctional [102]. This NGO-based multifunctional nanoplatform, nanohybrid could be used as a potential theranostic UCNPs-NGO/ZnPc, could be used as UCL high probe for upconversion luminescence (UCL) contrast imaging probing of cells and whole-body for imaging-guided combinatorial PDT/PTT. diagnosis, as well as for PDT causing the formation of cytotoxic 1O2 under light excitation and for PTT by 3.1.3. Imaging, drug/gene delivery, and photodynamic/ converting the 808 nm laser energy into thermal photothermal therapy energy. Another platform for combined PTT/PDT is The real potential of the graphene-based the one developed by Cho et al., in which nanoplatforms lies in the possibility of combining HA-conjugated Ce6 was combined with GO in order multiple strategies to fight cancer in a single platform. to improve biocompatibility [104]. The resulting As reported by Gazzi et al. [117], the opportunity to system (GO–HA–Ce6) was shown to be associate imaging diagnostic methods, drug delivery, enzyme-activatable, which could be used for both PTT with PDT opens the way to new approaches and NIR fluorescence imaging and photo-induced cancer enhances the efficacy of the single modalities. therapy. The following year another group combined A typical example of this enhancement is well PEG-functionalized GO with the PS 2-(1-hexyloxy- represented by the work of Feng L et al., where the ethyl)-2-divinyl pyropheophorbide-alpha (HPPH or authors fabricated a pH-responsive nanocarrier. NGO Photochlor®), via supramolecular π-π stacking [103]. was coated with two polymers, PEG and poly The system showed significant improvement in (allylamine hydrochloride) (PAH), modifying the photodynamic cancer cell killing efficacy due to the latter with 2,3-dimethyl maleic anhydride (DA) in increased tumor delivery of HPPH. Golavelli et al. order to acquire pH-dependent charge reversibility. developed a superparamagnetic graphene-based The nanocarrier was then loaded with DOX, to form nanoplatform; the MFGeSiNc4, an excellent the NGO-PEG-DA/DOX complex which showed T2-weighted MRI contrast probe [98]. The graphene responses to pH change, enhanced cellular uptake, NIR absorption ability (600-1200 nm) and the augmented DOX release in the tumor presence of silicon phthalocyanine bis microenvironment and inside cellular lysosomes. The (trihexylsilyloxide) (SiNc4) facilitated the slow efflux of DOX from NGO-PEG-DA/DOX offers immobilization of various PSs for the achievement of an enhanced killing of drug-resistant cancer cells both PTT and PDT effects using a single light source. compared with free DOX. Moreover, NGO-PEG-DA/ In vitro studies have suggested that MFG–SiNc4 may DOX has an excellent photothermal conversion thus be utilized as a potential theranostic nanocarrier ability; therefore, a synergistic therapeutic effect was for dual-modal imaging and phototherapy of cancer realized combining chemo- and PTT [118]. Similarly, a cells with a single light source for time and pH-responsive nanoplatform for controlled drug cost-effective treatments with a minimal therapy dose. release was developed by Battogtokh et al. where the In 2015, Kim et al. engineered ZnPc-PEG- author used a GO-based nanocarrier for Au@GON NPs, in which the PS zinc phthalocyanine pH-dependent release of the PS that was also used as was loaded onto PEGylated Au@GON. The system a fluorescent imaging agent [119]. showed promises for both combinational treatment of Five studies have used rGO as a starting material

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5449 for combined therapy and imaging [120–124]. For contrast agent to aid the delivery and pH- and example, Shervedani et al. developed, rGO-PDA- NIR-light dependent release of DOX in breast cancer BSA-DTPA-Mn(II)/MTX, a high biocompatible [127]. Another study using DOX as antitumoral drug system for breast cancer-selective PTT [120]. In this for combined drug delivery and PTT in an system, GO was partially reduced and functionalized MRI-capable nanomediator (rGO–Fe2O3@Au NPs) with dopamine to obtain a reduced graphene was reported by Chen et al. The superparamagnetic oxide/polydopamine system (rGO-PDA). As a highly nanoplatform showed a high photothermal promising carrier in drug delivery, bovine serum conversion efficiency (under 808 NIR laser albumin protein (BSA) was grafted onto the obtained irradiation) and an excellent drug loading ability in system (rGO-PDA-BSA). Finally, the researchers cervical cancer in vitro (HeLa cells) [124]. The delivery adopted the decoration with diethylenetriamine- of DOX in association with PTT was also evaluated by pentaacetic acid (DTPA)-Mn(II) to achieve diagnosis Khatun and co-workers [128]. Their graphene-based and methotrexate for anticancer therapy. nanosystem conjugated with a hyaluronic acid A new functionalization strategy was proposed, nanogel for photothermal imaging was able to induce where the photoresponsive imaging agent ICG has an effective killing of lung cancer cells (A549), while been loaded onto hyaluronic acid-anchored (HA) rGO showing only minor toxicity in the non-tumor MDCK nanosheets in order to enhance the photothermal cells. Jin and co-workers combined Au NPs into properties of the system. Compared to rGO, HArGO poly(lactic acid) microcapsules for combined drug resulted in enhanced tumor cell targeting in vitro and delivery and PTT in a GO-based nanosystem to in vivo [123]. Moreover, the ICG/HArGO targeted enhance ultrasound imaging and X-ray CT imaging delivery has been shown to guide authors to identify [115]. Thakur et al. developed a graphene-based the most relevant area for NIR irradiation to achieve nanoplatform for simultaneous imaging and PTT. Exploiting another functionalization strategy, a combined drug delivery, PTT, and PDT. The Gd-functionalized GO nanoplatfom for MRI guided nanosystem was able to perform as a NIR imaging photothermal-chemotherapy was developed [125]. agent and to induce the ablation on the tumor in vivo They have shown neglectable toxicity both in vitro and [60]. In 2014, Bian and collaborators fabricated in vivo studies, GO@Gd-PEG-FA was able to kill another graphene-isolated-Au-nanocrystal (GIAN) cancer cells selectively demonstrating to be an for multimodal cellular imaging by means of Raman excellent MRI guided photothermal-chemothera- scattering and NIR two-photons luminescence [129]. peutic system with drug delivery and tumor-targeting Besides having an exploitable DOX loading properties. In 2012 a new system characterized by capability, graphene-isolated-Au-nanocrystal (GIAN) biocompatibility, high DOX loading capacity, NIR showed NIR absorption that allowed using them also photothermal heating, facile magnetic separation, and for PTT. Using NIR heating, they obtained a large T2 relaxation rates (r2) was successfully controlled release of DOX, drastically reducing the developed [72]. A single system for image-guided possibility of side effects in chemotherapy. In the glioma therapy with integration of MRI, dual-modal same year, a GO-Au nanohybrid was fabricated by recognition (magnetic and receptor-mediated active chemical deposition of Ag NPs onto GO through a targeting), and chemo-photothermal therapy was hydrothermal reaction [106]. DOX was loaded to developed by Chen et al., based ongraphene-gold obtain an anticancer activity. Functionalization of nanohybrids, GO-Au@PANI, with excellent NIR GO@Ag-DOX nanohybrid with DSPE-PEG2000-NGR, photothermal transduction efficiency and ultrahigh resulted in GO@Ag-DOX-NGR, a particular drug-loading capacity [126]. By using this theranostic nanoplatform with powerful tumor- ultrasensitive nanoprobe, cancer cells were analyzed targeting capability, excellent stability in through SERS-fluorescence dual-mode imaging, physiological solutions and a much higher antitumor confirming optimal DOX-loading efficiency and efficacy without toxic responses owing to the higher delivery accompanied by an increased sensibility of DOX uptake at the tumor site. Moreover, GO@Ag– NIR/pH-responsive release. The GO-Au NPs DOX–NGR not only served as a diagnostic X-ray decorated with PANI, a new NIR PTT agent contrast probe, but also as a potential agent for chemo characterized by a strong NIR absorption, allowed the and photothermal therapy. GO@Ag–DOX–NGR was in vitro and in vivo chemo-photothermal ablation of demonstrated to have an ideal tumor-targeting breast cancer cells. Chang et al. produced a PEGylated capability with NIR laser-controlled drug release and GO/MnWO4 nanocomposite (GO/MnWO4/PEG) for X-ray imaging ability, ensuring a significant dual imaging (MRI and PAI) and therapy chemo-photothermal therapeutic efficacy. (chemotherapy and PTT). In vivo data demonstrated Three studies also explored GQDs for combined that the nanosystem was an excellent bimodal multiple therapies and imaging [121,130,131]. In the

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5450 first study, a porous silica NPs-based nanosystem, combined with the PS Ce6, both in vitro and in vivo encapsulated with the PS hypocrellin A and GQDs, data confirmed that efficient photoablation of tumors allowed combining multicolor imaging and cervical was achieved through the synergistic PDT and PTT cancer treatment [131]. In more recent work, effect under the activation of a single wavelength porphyrin derivatives were conjugated to an laser. aptamer-functionalized GQDs for miRNA delivery and simultaneous PTT /PDT, allowing CLSM of lung 3.1.4. Imaging and other new therapies and breast cancer cells [130]. Beyond drug delivery, PTT, and PDT, other Finally, concerning the simultaneous use of non-conventional therapies, like sonodynamic imaging, PTT, and drug delivery strategies, Chen and therapy (SDT) and immunotherapy, can also be co-workers used a valuable approach [121]. They enhanced by the use of graphene, GRMs, and fabricated biocompatible and photoluminescent graphene hybrid nanosystems. The following sections GQDs which, in conjunction with superparamagnetic give a short description of the recent advances of iron oxide nanoparticles, was exploited for both these materials used for these applications. fluorescent and MR imaging without the use of any i) Sonodynamic therapy (SDT). Ultrasound- other fluorescent dyes. Thanks to the external based therapies are opening new prospects in the magnetic stimulation, the drug has been continuously oncological field; amongst them, SDT emerged very and slowly released by the GQDs in vitro. The NIR recently as a novel cancer treatment approach. Being irradiation of HeLa cells and their consequent more effective than PDT due to the higher tissue photothermal ablation confirmed their potential use penetration depth [135,136], SDT consists in the use of for cancer PTT. Despite the few numbers of ultraviolet light as an external stimulus in order to publications, we can highlight three different studies activate a sonosensitizer, a non-toxic and selective reporting methods based on PDT combined with chemical agent that once activated is able to induce imaging and drug delivery [132–134]. In 2015, Yan et ROS synthesis and thermal effects. al. validated the effects of GO-PEG DVDMS for drug Despite nanoparticle-assisted ultrasound the- delivery, in vivo imaging, and PDT [132]. rapy being still under development in the clinical field Sinoporphyrin sodium (DVDMS), a novel [137], different cell death pathways involved in the photo-theranostic agent, was successfully loaded to SDT process have been identified. To this end, Dai et PEGylated GO via intramolecular charge transfer, al. have synthesised a novel nanoplatform for enhancing its fluorescent imaging and improving its MRI-guided SDT and PTT by the functionalization of tumor accumulation. In the same year, an MRI rGO with TiO2 NPs [135,138]. Graphene's high GO/MnFe2O4 nanohybrid was fabricated with very electrical conductivity enableds the separation of the low cytotoxicity and negligible in vivo toxicity [133]. sono-generated electron-hole pairs, leading to an MRI experiments demonstrated that the large increased in vitro ROS production. magnetic spin magnitude of manganese ferrite Recently, Huang et al. translated SDT into a (MnFe2O4) NPs make them suitable as reliable T2 theranostic context, by developing a graphene-based contrast enhancement agents. The GO/MnFe2O4 nanoplatform for PAI-guided SDT in breast cancer optical absorbance in the NIR region and the optimal cells and tissues, paving a new path toward targeted photothermal stability resulted in the highly effective medicine [139]. photothermal ablation of HeLa cancer cell lines. In ii) Immunotherapy. Immunotherapy is a new this study, nanohybrids were further tested for therapeutic approach that allows precise cancer chemotherapeutic purposes by combining with treatment enhancing or restoring the patients’ own DOX-induced chemotherapy. An enhancement in immune system's ability to fight cancer [140]. The cancer cell killing activity was achieved after introduction of 2DMs in these fields is aimed at GO/MnFe2O4/DOX irradiation with NIR light, when improving the patients’ outcome expanding the compared to free DOX. Finally, Wu et al. fabricated a combination of possible different therapeutic and graphene-Au nanostar hybridized system (denoted as imaging modalities in a single nanosystem. GO/AuNS-PEG) in order to achieve single For example, in 2014, GO was explored both for wavelength laser-induced synergistic PDT and PTT PTT and the delivery of CpG oligodeoxynucleotide and active cancer photothermal/fluorescence [141]. The latter can be recognized by Toll-like multimode imaging [134]. The system was shown to receptor 9 leading to the secretion of proinflammatory be biocompatible in vitro and in vivo. Under the NIR cytokines, resulting in the activation of innate and laser irradiation, authors achieved high adaptive immune responses [142]. The intracellular dual-enhanced photothermal efficiency, even for trafficking of nanocarriers was improved thanks to tumors found at deep locations. When the system was the local heating allowed by the optimal nanosystem

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NIR optical absorbance, leading to an efficient tumor light. The TMDs investigated so far in theranostics are reduction in vivo. tungstenum disulfide and molybdenum disulfide. Recently, Wang et al. developed a i) Tungsten disulfide. Tungsten disulfide has PEGylated-rGO nanoplatform hybridized with Fe3O4 received considerable attention in recent years, thanks nanoparticles for breast cancer dual treatment based to the fascinating physicochemical properties that the on PTT and immunotherapy [143]. Thanks to the whole family of TMDs have in common. WS2 is a incorporation of Fe3O4 nanoparticles, the resulting well-known solid lubricant for its tribological nanosystem induced the reduction of tumor- near-zero friction or superlubricity and possesses a associated macrophages and the activation of lamellar structure similar to that of MoS2, allowing it dendritic cells in tumor draining lymph nodes in to be exfoliated into nanosheets [146], which can be tumor-bearing mouse model. Moreover, the authors loaded with drugs as drug delivery carriers, exactly as envisage the application of their smart nanocomposite done for graphene. for MRI-guided therapy. However, only recently the The capability of WS2 to serve as a cancer first attempt to apply GRMs for immunotherapy in nanoteranostic tool mainly derives from its strong the field of cancer theranostics was made by Wu and absorbance in the NIR region and X-ray attenuation co-workers. In this study, polydopamine stabilized ability, making it a suitable photothermal and contrast GQD PSs were integrated with immunostimulatory agent for PAT and CT bioimaging-guided diagnosis polycationic polymer/CpG oligodeoxynucleotide and therapy. Exploiting its strong absorbance in the nanoparticles and Gd3+/Cy3 imaging probes, NIR region, Cheng et al. reported for the first time the enabling dual imaging (i.e., MRI and fluorescence opportunity of using WS2 nanosheets as novel agents imaging)-guided photoimmunotherapy [144]. The for PTT in association with multimodal bio-imaging, resulting theranostic nanostystem allowed the obtaining a highly effective photothermal ablation of effective eradication of tumor in a murine mammary tumor in a mouse model [147]. Their PEGylated WS2 cancer model, because of the combination of nanosheets enabled an excellent NIR light-triggered enhanced PDT and PTT exerted by GQDs, together tumor ablation after both intratumorally (low dose, 2 with the simultaneous activation of endosomal mg/kg) and intravenous injection (high dose, 20 Toll-like receptor 9 mediated by the polycationic mg/kg). Moreover, PEG-WS2 nanosheets demon- polymer/CpG oligodeoxynucleotide. This immuno- strated to serve as a bimodal contrast agent for CT and stimulation resulted in the secretion of PAT imaging, due to their strong X-ray attenuation proinflammatory cytokines and dendritic cell ability and the high NIR optical absorbance, maturation, ultimately triggering the activation and respectively. infiltration of T cells. Another example of WS2 nanosheets-based theranostic application is represented by the work of 3.2. 2D materials beyond graphene for cancer Yong et al. [148]. In this case, the developed WS2 theranostics nanosheets were employed not only as NIR absorbing After the rise of graphene, the whole family of agents for PTT, but also as PS carriers for PDT. 2DMs has started to be investigated for several Moreover, the PSs release behavior from WS2 applications, including the fight of cancer. GDY and nanosheets could be controlled by NIR irradiation 1 other emerging 2DMs, such as WS2, MoS2, hBN, BP, manipulating O2 generation of the PSs-WS2 complex. and MXene, are under consideration for their use as WS2 was used for theranostic purposes as cancer theranostic tools [145]. All the reports present reported by the work of Yang et al., where IONPs in the literature for these materials as theranostics are were adsorbed on PEGylated WS2 nanoflakes, which discussed and reported in Table 4. The following were subsequently coated with silica and manganese sections are dedicated to each type of material. dioxide [149]. The resulting nanoplatform (WS2-IO/ 3.2.1. The 2D family of transition metal S@MO-PEG) appeared to be highly sensitive to pH, enabling tumor pH-responsive MR imaging using dichalcogenides IONPs and MnO2 as pH-inert T2 contrast probe and TMDs have the empirical formula MX2; where M pH-sensitive T1 contrast probe, respectively. is a Group 6 transition metal (usually Mo or W) and X WS2-IO/S@MO-PEG allowed the synergistic is a group 16 calcogen (S, Se or Te). In bulk form, combination of NIR light and X-ray absorbance of TMDs are layered compounds which can be WS2 for PTT and cancer radiotherapy, resulting in exfoliated down to few and single layers. In remarkable tumor destruction. particular, in the single-layer form, many TMDs show Furthermore, with regards to cancer strong light-matter interaction, i.e., strong absorption radiotherapy, Chao et al. have recently tested a of light in the visible-near IR range and emission of WS2-based theranostic nanosystem functionalized

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188 with PEG and labeled with Re, a widely-used MoS2/Fe3O4-PEG (MSIOs) composite consisting of radioisotope for radioisotope therapy (RIT) [150]. The NIR-absorbing MoS2 flakes for PTT decorated with 188 Re labeling of WS2-PEG nanoflakes enabled not Fe3O4 nanoparticles as target moieties spatially/ only the enhancement of RIT, but also allowed the in timely guided by an external magnetic field to cancer vivo tracking of the nanoflakes after their tissue [156]. Thanks to their superparamagnetic administration into animals. property and high NIR absorption, MSIOs ii) Molybdenum disulfide. Molybdenum nanosystem for magnetic targeted PTT was applied disulfide is another relevant member of the 2D family for MR and PAT imaging in vitro for cervical (HeLa of TMDCs, with properties very similar to WS2. Thus, cells) and liver cancer (HepG2 cells). Soon after, in it has been investigated as a theranostic cancer 2016, Li et al. synthesized biocompatible soybean nanoplatform with multiple highly integrated phospholipid-encapsulated MoS2 (SP-MoS2) nano- functionalities [151]. In particular, it has been sheets for in vitro and in vivo highly efficient breast explored as a photothermal agent for cancer PTT and cancer PTT using a laser with wavelengths of 808 nm as a nanocarrier for drug delivery-based therapeutic and IR thermal imaging [157]. The encapsulation of approaches. The principal limit for MoS2 application the nanosystem with soybean phospholipids was in biomedicine appears to be mainly related to its low used as an alternative to PEG functionalization to stability in the biological milieu [152]. Therefore, in enhance its colloidal stability, allowing improving the the majority of the studies, different water-soluble therapeutic performance. The following study aimed and biocompatible molecules were applied to at exploiting MoS2 proprieties for cancer theranostics functionalize MoS2 to overcome this issue. In 2014, Liu was based on a different kind of material synthesis, et al. have reported the first demonstration for the use where polyvinylpyrrolidone (PVP) was used not only of MoS2 in cancer theranostics, modifying the material to enhance the colloidal stability but also to direct the to obtain a high biocompatible PEGylated MoS2 growth of MoS2 nanosheets to produce an ultra-small loaded with the photodynamic agent Ce6 MoS2-PVP composite for in vitro and in vivo combined (MoS2-PEG/Ce6). The nanocarrier was able to PTT and PAI [158]. enhance the intracellular delivery of Ce6, dramatically In 2017, Xu et al. integrated MoS2 nanosheets increasing the PDT efficacy in vitro in breast cancer with IR-808 sensitized upconversion nanoparticle cells (471 cell line) exposed to 660 nm light irradiation. (UCNP) loaded with Ce6 for synergistic PTT and PDT Moreover, it further increased after induction of a in vitro and in vivo, simultaneously achieving trimodal moderate hyperthermia (808 nm laser irradiation) to UCL, CT, and MR imaging under a single 808 nm promote the cellular uptake. Moreover, both in vitro wavelength laser excitation [159]. Similarly, Liu et al. and in vivo data demonstrated the ability of designed a trimodal imaging (IR, PA, and MRI) MoS2-PEG/Ce6 to promote tumor ablation exploiting Mo@Fe-ICG/Pt multifunctional nanocomposite, a combined PTT and PDT synergistic affect triggered consisting of polyethyleneimine (PEI) functionalized by laser with wavelengths of 808 nm and 660 nm, MoS2 nanosheets ingeniously decorated with Fe3O4 respectively. Also, the PEGylated MoS2 nanosheets nanoparticles and loaded with ICG molecules and served as a contrast agent for photoacoustic platinum (IV) as PSs and prodrugs, respectively [160]. tomography (PAT) allowing the in vivo tracking of the The resulting nanosystem showed a remarkable nanosystem, and the effective photothermal heating tumor cell killing ability both in vitro and in vivo by was confirmed by IR thermal imaging [153]. taking advantages of the synergistic PTT, PDT, and In their subsequent study, in addition to PAT, chemotherapy triggered by a single 808 nm NIR laser. the imaging was also performed in association to MR In the same year, Cheng at al. conjugated bovine and PET in mice, thanks to the superparamagnetic serum albumin-gadolinium (BSA-Gd) complexes with properties of PEGylated MoS2 decorated with iron MoS2 nanoflakes (MoS2-Gd-BSA) for PTT and oxide (MoS2-IO-PEG) and to the adsorption of the bimodal MR and PA imaging [161]. The 64 positron-emitting radioisotope Cu, respectively. The MoS2-Gd-BSA biocompatible nanosystems presented triple-modal imaging-guided tumor PTT by the 808 excellent tumor cell inhibition effectiveness in vitro nm laser resulted in the complete elimination of 471 and could totally ablate cancer in vivo using 808 nm breast cancer tumors in vivo [154]. In the same year laser irradiation, without any sign of recurrence in the (2015), Wang et al. produced a MoS2/Bi2S3-PEG (MBP) next two weeks of follow up. Maji at al. synthesized a composite, with enhanced colloidal stability and hybrid gold nanobipyramid nanostructure coated biocompatibility, for PTT combined with with MoS2 (AuNBPs@MoS2) for enhanced photoacoustic and CT imaging, demonstrating its photothermal conversion and ROS production-based therapeutic efficacy in vitro and tumor-bearing 4T1 cancer treatment and simultaneous two-photon mice [155]. In the meantime, Yu at al. created luminescence imaging in HeLa cells [162]. Most recent

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5453 works explored a therapeutic approach based on drug after material injection resulted in photothermal delivery to counteract drug-resistant cancer. In 2018, ablation of tumor [170]. Later, other studies used Dong et al. used a biodegradable hyaluronic acid (HA) PEGylated BP nanoparticles to improve their and PEI-decorated MoS2 nanocarrier (MoS2-PEI-HA) anti-cancer activity in theranostics by combining with loaded with DOX [163]. HA was used as a targeting AuNPs [171] or chemotherapeutic drug DOX [172]. moiety against CD44-overexpressing breast cancer Among the attempts to improve imaging properties, cells (MCF-7-ADR) and, thanks to its localized BP has been conjugated to other nanoplatforms for biodegradation by hyaluronidase concentrated at the MR or PAI [173–177]. tumor site, it also served for controlled and faster DOX release. Moreover, the composite was labeled 3.2.3. MXenes (carbides and nitrides) with 64Cu for PET imaging in vitro and in vivo. MXenes are among the most recently discovered Another study also exploited drug delivery through 2DMs [178]. The term MXenes comprehensively the loading of chitosan and metformin on Mn-doped defines early-transition-metal carbides, carbonitrides, Fe3O4@MoS2 composites for combined PTT and MR and nitrides characterized by structural formula imaging [164]. In vitro data demonstrated that the load Mn+1Xn, where M is an early transition metal, X is with metformin led to hepatic cancer ablation (Hep3B carbon, nitrogen, or both, and n = 1-3. MXenes are cells) not affecting healthy cell viability (LO2 cells), synthesized by selective etching the A-group element while chitosan, besides enhancing the dispersibility from the precursor ternary-layered carbides of MAX and biocompatibility of the nanoplatform, and was phases, where A is a group 12−16 elements of the able to improve the PTT efficiency. Finally, a similar periodic table (Figure 2A-C). MXenes contain approach was employed in a very recent publication abundant surface-terminating functional groups, such concerning the use of MoS2 for cancer theranostics as hydroxyl (−OH), oxygen (−O), or fluorine (−F), [165]. In this work, hyaluronic acid which endow them with a hydrophilic nature and (HA)-functionalized MoS2 nanosystems were applied allow flexible surface modification, functionalization, to deliver Gd and the anticancer drug gefitinib (Gef) and scalable processability. MXenes share a number for combined chemo-PPT and MR. This smart of the advantageous properties of 2DMs for nanoplatform induced the inhibition of lung cancer in application in biomedicine stemming from their vitro and in vivo. topology, including extreme thinness, high surface area, high surface-to-volume ratio, and mechanical 3.2.2. Black phosphorus toughness. Furthermore, they show remarkably high Phosphorous is one of the most abundant volumetric capacitance (1,500 F/cm3) [179] and elements on Earth and can exist in various allotropes, metallic conductivity (~10,000 S/cm) [180], and rich namely white, red, black, and violet phosphorus. Bulk surface chemistry for enzyme or drug black phosphorus (BP), the thermodynamically most functionalization. The field of cancer theranostics, stable allotrope of phosphorus [166], is one of the however, is undoubtedly spearheading the field of emerging monoelemental materials, which have biomedical applications of MXenes as they are shown outstanding potential in biomedical increasingly attracting attention as PTAs for applications [167,168]. It is a semiconductor 2DM with synergistic chemo/photothermal (PTT)/PDT and a tunable bandgap dependent on its thickness. In the imaging, due to the early recognition of outstanding single-layer form, each phosphorus atom is covalently internal photothermal conversion efficiency and light bonded with the P atom on both sides of the same absorption capability [181]. plane, and the other P atom is in the neighboring Han et al. demonstrated a platform for PTT, plane through the 3p orbitals. BP actively interacts chemotherapy, and imaging-based on Ti3C2 with incident light, owing to its tunable bandgap nanosheets (NSs, Figure 2C) functionalized with ranging from UV to NIR, depending on the thickness. soybean phospholipids (SP), which significantly Thus, this opens the door for its possible applications improved the colloidal stability of Ti3C2 NSs in a in various fields, including bio-photonics, physiological environment without affecting their photocatalysis, and bioimaging [169]. In one of the photothermal properties (Figure 2A) [182]. earliest theranostic studies with BP, Sun et al. used UV-VIS-NIR absorption spectroscopy on aqueous PEGylated BP nanopartcles in order to have better Ti3C2 solutions showed a characteristic spectrum with stability (single and few-layers BP are unstable in the peak absorption at ~800 nm, right within the first NIR air), biocompatibility and long-blood circulation. absorption window. The abundant OH- surface Following PAI in vivo, BP nanoparticles were shown terminations on in Ti3C2-SP MXenes were used to to accumulate in through the enhanced permeability adsorb electrostatically cationic molecules such as retention effect. NIR light irradiation of these tumors DOX (Figure 2A) and achieve a drug-loading

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capability of up to 211%. Once the Ti3C2-SP-DOX NSs enhanced-contrast in situ CT imaging, MnOx or IONP penetrated inside 4T1 breast cancer cells, DOX release were the contrast agents for MRI and Ta4C3 was the was triggered endogenously by the acidic tumor high-efficiency PTA for PAI and PTT. microenvironment through the interference of H+ The possibility of working with PTA with high ions with the electrostatic interactions between DOX conversion efficiency also in the NIR II biowindow and Ti3C2-SP, or exogenously via NIR irradiation (1000-1300 nm) offers several advantages, including (Figure 2A). Compared to single therapeutic higher tissue penetration depth and maximum -2 modalities, Ti3C2-SP-DOX synergistic chemotherapy permissible exposure limit (1 W cm ) compared to 808 and PTT led to enhanced inhibition of 4T1 cancer cell nm (0.33 W cm-2, ANSI Z136.1−2007, American viability in vitro as well as to improved therapeutic National Standard for Safe Use of Lasers). Compared and recurrence outcomes in vivo in 4T1 breast to Ti3C2 and Ta4C3, Nb2C MXene shows a strong and cancer-bearing mice. Furthermore, Ti3C2-SP showed almost constant optical absorption in the NIR I and significant contrast-enhancement for intratumor PAI NIR II biowindows [189] (Figure 2G). Taking up to 24h post-injection. In a similar system, Liu et al. advantage of this favorable broadband absorption, first demonstrated the feasibility of Ti3C2 NSs as PSs Lin et al. demonstrated a theranostic platform for PTT for PDT in a synergistic platform for and PAI in 4T1 breast cancer cells based on Nb2C NSs PTT/PDT/chemotherapy. The Ti3C2 NSs-mediated stabilized with biocompatible polyvinylpyrrolidone generation of reactive oxygen species (ROS) was (PVP) with photothermal conversion efficiencies of investigated with 1,3-diphenyl-sobenzofuran (DPBF) 36.5% at 808 nm and 46.65% at 1064 nm, respectively 1 as the singlet oxygen ( O2) detector. NIR irradiation of [189]. In the attempt to extend the optical absorbance Ti3C2 NSs at 808 nm for 10 min, led to a ~80% decrease range of Ti3C2, Tang et al. proposed a Ti3C2-Au in DPBF absorbance at 420 nm, thus revealing the nanocomposite synthesized by seed-growing Au on 1 generation of O2. ROS production, although less the surface of the Ti3C2 NSs (Figure 2H), followed by pronounced, was also observed when Ti3C2-DOX NSs PEGylation to achieve colloidal stability. The Au were exposed to the same irradiation protocol [183]. decorations significantly enhanced the photothermal 1 Proposed mechanisms for O2 generation in Ti3C2 conversion efficiency compared to Ti3C2 NSs of both attributes it to the energy transfer of photoexcited at 808 nm (34.3% Vs. 30.7%) and 1064 nm (39.6 Vs. electrons from Ti3C2 to triplet oxygen (ground state 28.3%, Figure 2I). Furthermore, the strong X-ray 1 oxygen, O2), similar to the photodynamic behavior of absorption capability enabled simultaneous CT and graphene quantum dots [184] and black phosphorous PAI up to 24-h post-injection (Figure 2J), as well as a [185]. Synergistic PTT/PDT/chemotherapy with synergistic PTT/radiotherapy platform. Interestingly, Ti3C2-DOX led to significant improvements in synergistic PTT/ radiotherapy appeared to most therapeutic efficacy and recurrence outcomes against effectively inhibit breast cancer growth in vivo human colon carcinoma (HCT-116) in vivo in compared to PTT or radiotherapy alone [190]. tumor-bearing mice. The abundant surface Possible challenges when engineering 2DMs as terminations in the Ti3C2 NSs also enabled drug-delivery carriers are the lack of a confined space functionalization for specific tumor targeting, such as for high loading of drugs, as well as achieving optimal hyaluronic acid coatings which increased colloidal drug release profiles. To enhance drug stability and enabled active targeting of the surface loading/release capabilities and tumor targeting Li et protein CD44+ overexpressed in cancer cells. al. surface-engineered Ti3C2 MXene via sol-gel To expand the specific tumor-imaging chemistry to create a mesoporous silica shell directly capabilities and realize a theranostic platform on the NSs (Ti3C2@mMSNs) using cetanecyltrimethyl- integrating multimodal imaging, such as PA, ammonium chloride (CTAC) as the mesopore- magnetic resonance (MR) and X-ray computerized directing agent and tetraethylorthosilicate (TEOS) as tomography (CT) imaging (Figure 2D) it is possible to the silica precursor in an alkaline environment. The substitute Ti in the M-phase with Ta, a transition resulting Ti3C2@mMSNs showed a uniform structure metal with higher atomic number (Z=73) and X-ray with high pore volume (0.96 cm3 g−1) with an average attenuation coefficient (4.3 cm2 g-1 at 100 keV) [186]. size of 3.1 nm. The NSs were also functionalized with Ta4C3 MXene displays concentration-dependent arginine-glycine-aspartic acid (RGD) for specific broadband light absorption in the UV-VIS-NIR tumor targeting and DOX for chemotherapy. Drug [187,188] and high photothermal conversion efficiency delivery through the Ti3C2@mMSNs was triggered via (>30%). Ta4C3-SP MXene functionalized with MnOx combined pH modulation, with an enhanced release [187] or superparamagnetic iron-oxide nanoparticles in an acidic environment, and local hyperthermia (IONP, Figure 2E-F) [188] realized a multimodal through NIR irradiation [191]. As the positively- imaging and therapeutic platform where Ta enabled charged surfactant, CTAC shows chemotherapeutic

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5455 effects, Han et al. [192] proposed a similar (CTAC@ Nb2C-MSN) and functionalized with RGD surface-engineering scheme based on 2D Nb2C (Figure 2K). MXenes coated with mesoporous silica shells

Figure 2. Illustration of MXene properties. (A) Schematic representation of Ti3C2 MXene NSs synthesis, functionalization with SP and DOX for colloidal stability and multistimuli-triggered chemotherapy, and illustration of a synergistic platform for cancer therapy based on Ti3C2 MXene NSs. (B) Scanning electron microscopy (SEM) of Ti3C2 after HF etching of the A phase and (C) transmission electron microscopy (TEM) of Ti3C2 NSs after exfoliation. Adapted with permission from [182], copyright 2018 Advanced Healthcare Materials. (D) Dual-mode CT/MR imaging contrast-enhanced by Ta4C3. (E, F) TEM of Ta4C3 (E) before and (F) functionalization with IONPs. Adapted with permission from [188], copyright 2018 ACS Nano. (G) UV-VIS-NIR absorption spectra of Nb2C MXene NSs at different concentrations. Adapted with permission from [189], copyright 2018 Journal of the American Chemical Society. (H) TEM of Ti3C2@Au nanocomposites. (I) UV-VIS-NIR absorption spectra of Ti3C2@Au-PEG at varying Au/Ti ratios. (J) PAI enhancement with Ti3C2@Au compared to Ti3C2 at the same Ti3C2 concentration (0.5 mg/mL). Adapted with permission from [190], 2018 ACS Nano. (K) High-resolution SEM of CTAC@ Nb2C-MSN NSs. Reproduced with permission from [192], copyright 2018 Theranostics. (L) Schematic illustration of free-radical generation with AIPH@Nb2C@mSiO2 nanocomposites under NIR irradiation at 1064 nm. (M) Photo-thermodynamic free-radical generation with AIPH@Nb2C@mSiO2 in 4T1 breast cancer cells detected by dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence under normoxic and hypoxic conditions. Adapted with permission [193] from 2019 ACS Nano.

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Due to the mesoporous silica reservoir and the (EPR) effect, without evidence of accumulation in the broadband absorption of Nb2C NSs, the CTAC@ major organs. Additional works report no sign of Nb2C-MSN display a drug loading capacity of 32.7% Ti3C2 NSs-induced red blood cell hemolysis in vitro and photo-triggered release upon irradiation at 1064 [196], and similar findings have been reported for the nm, although the photothermal conversion efficiency cyto- and systemic biocompatibility for Ta4C3 appears to be lower than unmodified Nb2C-PVP NSs [187,188] and Nb2C MXenes [189]. [189]. Feasibility of CTAC@ Nb2C-MSN as PTA in a Finally, controlling the lifetime of the nanoagents synergistic PTT-chemotherapy and PAI theranostic in the body and mitigating the risks related to platform was demonstrated against U87 glioblastoma retention of nanomaterials and their byproducts, cell lines in vitro and in vivo on glioblastoma-bearing could significantly advance nanomaterial-based mice. theranostic platforms in the translational pipeline. Effective generation of ROS is critical for PDT. Recently, an active biodegradation scheme for Nb2C ROS generation is controlled by local oxygen tensions, MXene NSs has been proposed [189]. This process which affects PDT efficiency in the hypoxic tumor leverages human myeloperoxidase (hMPO), a microenvironment. To circumvent this issue, Xiang et free-radical species generating enzyme expressed by al. developed an alternative “thermodynamic” cancer neutrophils to carry out their antimicrobial activity. In treatment strategy based on the thermally-activated the presence of H2O2, hMPO generates hypochlorous free-radical generator 2, 2′-Azobis[2-(2-imidazolin- acid and reactive radical intermediates, which 2-yl)propane] dihydrochloride (AIPH) loaded into the degrade polymers and carbon-based materials. The mesopores on a silica-coated Nb2C MXene incubation of Nb2C NSs in hMPO and H2O2 enriched (AIPH@Nb2C@mSiO2, Figure 2L). AIPH is a medium for 24 h caused the complete degradation hydrophilic and thermo-labile molecule that can and disappearance of NSs, thus demonstrating in vitro generate ROS independent of oxygen concentration. the feasibility of this enzyme-triggered degradation Therapeutic efficacy of the synergistic PTT and route for MXenes. free-radical generation via hyperthermia under NIR-II irradiation was demonstrated in vitro in both 3.2.4. Other emerging 2D materials: graphdiyne, normoxic and hypoxic conditions (Figure 2M) and in hexagonal boron nitride, silicene, antimonene, vivo, resulting in complete tumor eradication in breast germanene, biotite, metal-organic frameworks, and layered double hydroxides cancer-bearing mice. AIPH@Nb2C@mSiO2 was also demonstrated to be an effective multimodal imaging Beside the abovementioned 2DMs, it is worth agent for real-time monitoring of the cancer considering other kind of emerging 2DMs with strong therapeutic process via PAI, fluorescence and potential in the field of cancer theranostics. photothermal imaging [193]. Considering the large number of 2DMs available, we The results obtained thus far on MXene-based have considered new 2DMs that are representative of theranostic platforms are encouraging. However, the less than the 6% of the works present into the field is still in its infancy and there is extensive work literature: graphdiyne, hexagonal boron nitride, some to be done in order to fully realize the potential of members of the Xenes family such as silicene, MXenes as nanoagents for multimodal cancer therapy antimonene (AM), and germanene [167], biotite, and imaging. Future work is required to elucidate the metal-organic frameworks (MOFs), and layered fundamental principles controlling the photothermal double hydroxides (LDHs). conversion and ROS generation activity of MXenes, as i) Graphdiyne. Graphdiyne is a carbon allotrope well as to fully expand and explore the capabilities of made of a combination of sp- and sp2-hybridized the different species available in the library of carbon atoms, which was first synthesized in 2010 MXenes. Most importantly, the long-term safety [198]. This particular structure gives these material profiles of MXenes need to be thoroughly and different physicochemical properties when compared extensively elucidated. They are being the first to graphene, which is only formed by sp2-bonded discovered and the most extensively characterized, carbons. It is characterized by uniformly distributed many of the preliminary studies available focus on subnanometer pores, making GDY a potential Ti3C2 biocompatibility. To date, there is no reported candidate for applications in the area of clean energy evidence of apoptosis or signs of cytotoxicity in vitro as a new system for gas separation [199]. From a on cancer cells [182,194–196] and cultured neurons biomedical point of view, GDY shows outstanding [197] from Ti3C2 exposure. Ti3C2 NSs injected in vivo in drug loading efficiency and high photothermal the bloodstream appear to be either excreted in the conversion ability, making it a promising tool for urine via physiologic renal clearance or retained in the tumor-fighting. In this view, few recent works have tumor via the enhanced permeability and retention started to explore its application for cancer treatment,

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5457 such as the study of Jin et al., which applied GDY as a (II) phthalocyanine (CuPc). CuPc molecule in this dual therapy platform for PTT and combined drug construct plays a double role as a PS for PDT as well delivery of DOX in vitro and in mice [200]. A very as a diagnostic tool for in situ monitoring and imaging recent study of Li et al. also designed a specific GDY of miR‐21 by surface‐enhanced Raman spectroscopy nanoplatform for cancer diagnosis as a (SERS). miR-21 was selected as a target miRNA with photoelectrochemical biosensor for ultralow levels of expression levels significantly elevated in a variety of cancer marker detection (microRNA let-7a) in human cancers, especially in breast cancer [214]. The main serum [201]. In 2017, GDY was introduced in cancer reason for using h-BN nanosheets in this composite theranostics as photothermal-acoustic wave was to improve the SERS signal obtained from CuPc nanotransducer for simultaneous PTT and PAI in vivo molecule. Interestingly, Raman signal of CuPc was - this is the only study published in the field so far, to negligible on the blank SiO2 while remarkable on h‐ the best of our knowledge [202]. Thanks to the BN nanosheets and higher than the one on other PEGylation-enhanced biocompatibility, GDY was 2DMs with the same number of layers, such as simultaneously exploited as a safe PTT agent and a graphene and MoS2. The SERS signal enhancement PAI probe. Owing to the high extinction coefficient in was attributed to the interface dipole interaction the NIR region and the excellent photothermal between h‐BN nanosheets and CuPc induced by the conversion efficiency (42%), the nanoplatform highly polar B−N bond. Moreover, the intensity of showed an outstanding photoacoustic response and CuPc was independent of the h‐BNNS thickness. Both an ideal photothermal capability, exhibiting an in vitro and in vivo data demonstrated that this efficient photothermal ablation of tumor in vivo. nanoplatform accumulates efficiently in tumor sites ii) Hexagonal boron nitride. Structurally, and has a remarkable therapeutic effect with single-layer hexagonal boron nitride is an analog of minimized damage to healthy tissues [215]. graphene, where B and N atoms are bonded to form a iii) Silicene. Silicene has attracted increasing hexagonal lattice. Despite the structural similarity, attention in the theranostic field. It belongs to the h-BN has very different properties compared to family of Xenes, which are 2DMs with X being Si, Ge, graphene: it is an insulator and has exceptional Sn, Sb, etc. This crystal is characterized by a hexagonal chemical and thermal stability, which make it an ideal honeycomb lattice presenting a non-planar buckled encapsulating layer in 2DMs based devices [203,204]. configuration. Thanks to its dimensionaliy, silicene In the biomedical field, a great deal of studies focused stands out due to its oustanding properties, which on applications of boron nitride nanotubes, since their include superconductivity [216], quantum spin Hall biocompatibility profile was more favorable magnetoresistance [217,218] and chirality [219], to compared to carbon nanotube counterparts [205,206]. name a few examples. In a recent work in cancer Thus, the interest in h-BN nanosheets for biomedical theranostics, silicene was used as biodegradable applications has emerged only in recent years, driven phototherapeutic agent [220]. In particular, the by the availability of highly concentrated h-BN authors functionalized silicene with bovine serum dispersions in water [207]. Yang et al. [208] first albumin (SNSs-BSA) for cancer PTT and PAI of mice reported exfoliation of h-BN using pyrene derivatives tumor xenografts. In vivo data demonstrated the in order to synthesize water dispersible and stable efficiency of SNSs-BSA-based cancer PTT, due to the dispersions of h-BN. The method was further high photothermal conversion performances showed improved by McManus et al., which demonstrated by the nanosystem and its resulting degradation highly concentrated dispersions with excellent accelerated by the photothermal activity. These biocompatibility profile in different cell lines up to a outstanding properties, along with SNSs-BSA concentration of 100 µg/mL [209]. Proof of concept extraordinary photoacoustic contrast as well as studies reported the potential application of h-BN as a intrinsic ambient degradability and biocompatibility, vector for the delivery of chemotherapeutic agents make silicene a promising multifunctional (e.g., DOX), oligonucleotides for immunotherapeutic nanoplatform for photo-triggered therapeutics and purposes in cancers, and, recently, for the combined diagnostic imaging. PTT and chemotherapy [210–213]. Application of iv) Antimonene. Antimonene is a member of the h-BN nanosheets in the field of theranostics is still in Xenes with X=Sb, which attracted strong interest in its infancy, with only one work proposing h-BN for the community due to its changes in electrical theranostic applications [214] so far, to the best of our properties with decreasing thickness, from semimetal knowledge. In detail, Liu et al. developed a into semiconductor at the single layer [221,222]. multifunctional CuPc@HG@BN theranostic platform Indeed, AM is characterized by high thermal composed of hexagonal boron nitride nanosheets (h‐ conductivity, superior stability, excellent carrier BNNS), conjugated DNA oligonucleotide, and copper mobility, good strain-induced band transition, and

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5458 unexpected spintronic properties [221–224]. theranostic system emerged as an intelligent tool with In a recent study by Tao et al., antimonene was unique features. The BM-PEG NSs can be activated by studied as a photothermal agent for minimal invasive a 650 nm laser, in order to produce anion superoxide and high selective PTT [225]. The strategy adopted by ·O2- from molecular oxygen (O2), or by a 808 nm laser the authors relied on the application of novel 2D AM to induce local hyperthermia. Moreover, PAI as well quantum dots for cancer treatment. The nanosystem, as fluorescent and photothermal imaging capabilities synthesized by liquid exfoliation method and of the engineered nanosystem enabled the functionalised with PEG to enhance its multimodal imaging-guided breast cancer treatment. biocompatibility and stability, exhibited a rapid vii) Metal-organic frameworks. These materials NIR-triggered degradability and a noteworthy are composed by coordinated metal cations or NIR-triggered tumor ablation in vitro and in vivo. clusters, which are held together by organic bridging Encouraged by these promising results, in a more ligands, creating a porous structure [230–234]. recent study, the same group further explored AM as Typically, MOFs have a 3D structure, but they can an innovative photonic drug-delivery nanosystem for also be made in a layered form, bridging the field of cancer theranostics [223]. PEGylated AM nanosheets 2DMs. In the theranostic field these materials have have shown superior accumulation and penetration at shown exceptional promise [8]. tumor sites, but to their extraordinary photothermal For example, in a recent study, Zhao et al. tested properties, excellent DOX-loading capacity, a new theranostic MOF-based nanoplatform, where controlled NIR light- and pH-triggered drug release, the material is used as both magnetic fully metabolic degradability, as well as resonance-contrast agent and DOX carrier, enabling multimodal-imaging properties (i.e., PAI/fluores- an improved therapeutic outcome compared to the cence imaging and PT imaging). The combination of one obtained with DOX alone [235]. Following the these outstanding properties resulted in a noteworthy same path, another group designed a MOF-based inhibition of tumor growth in vivo without recorded theranostic Fe3O4@UiO-66 core–shell for combined side effects and optimal clearance of the nanoplatform MRI and drug delivery [236], obtaining tumor from the mouse body, suggesting promising eradication in both in vitro and in vivo studies. applications in the field of cancer theranostics. Another MOF-based tumor targeting drug delivery v) Germanene. Germanene belongs to the Xenes system with fluorescent properties was developed by family and shows promising properties for Gao et al. allowing the guided-MRI of cancer [237]. application in electronics [226,227]. In the theranostic Further works regarding the application of field, an innovative study has been successfully imaging-trackable MOFs into PDT [238,239] and PTT performed by Ouyang et al., leading to the [240] were carried out with positive results, exploring development of functionalized 2D germanene also MRI as a diagnostic modality combined with quantum dots (GeQDs) [228]. GeQDs were assessed to multiple therapies (i.e., drug delivery and PTT) in a be excellent PTAs owning an outstanding single MOF-based system [241]. photothermal conversion efficacy (superior to viii) Layered double hydroxides. 2D structured graphene and BP-based QDs) [225], higher stability layered double hydroxides have aroused extensive [167], and optimal biocompatibility [225]. Moreover, curiosity in the scientific community. New in order to define a complete multimodal nanosystem intercalative nanohybrids, such as inorganic-, organic- for cancer theranostics, beside the therapeutical and bio-LDHs, have displayed extremely synergetic aspects, GeQDs were proved to be useful also for properties and complementary performances, being multimodal diagnostic imaging (fluorescence/PAI used first as antacid and anti-pepsin agent named /photothermal imaging-guided hyperpyrexia “Talcid®” (Bayer) [242] and then as a biocompatible ablation of tumors). carrier/reservoir for drug and gene delivery [243]. vi) Biotite. Biotite, or black mica (BM), consists LDH is a class of inorganic 2D nanolayers [244,245] of sandwiched sheets of silicate minerals offering and one of the most commonly used nano-carriers in great potential in the theranostic field. The unique drug delivery systems. In the theranostic field, structure of BM lays the foundations for the design of LDH-based nanohybrids, found a place for a multiple ROS-mediated combined PDT and multimodal imaging in combination with different chemodynamic therapy (CDT). Indeed, the BM-PEG anticancer therapeutic approaches in order to realize NSs recently introduced by Ji et al. [229] served as synergistic treatments. LDHs have been widely robust theranostic nanoplatform for multimodal studied as delivery carriers for theranostic approaches imaging-guided chemodynamic therapy, PDT and in different therapies, including drug and gene PTT. Thanks to the presence of MgO, Fe2O3, and FeO delivery, phototherapy and even immunotherapy in the PEGylated BM structure, this new engineered [246–251]. Following this line of research, Wang et al.

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[246] developed a Gd-doped layered double In the case of 2DMs, the importance of hydroxide (LDH)/Au nanocomposite for cancer characterization has been recognized as a high bimodal imaging of and drug delivery. The new priority by the community [253–256]: this is because nanocomposite was shown to have a high DOX different forms of graphene-based materials - each of loading capacity and an interesting pH-responsive them with its own properties - can be made, drug-release profile. Moreover, the nanosystem depending on the production method and processing demonstrated a high performance in vitro and in vivo used. For example, using graphene oxide provided by CT and T(1)-weighted MRI attitude without different commercial sources may give different cytotoxicity or tissue damage. Another pH-responsive results because it is produced using different nanoplatform was tested by Huang et al. [247]. The protocols, giving rise to materials with a different new MnFe-LDH releases paramagnetic Mn2+ and Fe3+ structure (e.g., C/O content), which will behave ions when in contact with the acidic microenviron- differently in the biological environment (see stability, ment of solid tumors producing the enhancement of degree of functionalization, etc). A change in the the T1 MRI contrast. In addition, the layered structure exfoliation parameters used to produce graphene by enables the delivery of chemotherapeutic drugs in a liquid-phase exfoliation can provide graphene with pH-controlled manner, and therefore it can different size, thickness or defects concentration [256]. simultaneously inhibit the growth of solid tumors. Similar issues can be extended to other 2DMs. Thus, it Interestingly, Peng et al. [250] studied a is of primary importance to determine the nature of DOX&ICG/MLDH composite material showing both the material produced and how this changes during pH-controlled and NIR-irradiation-induced DOX the processing. In the case of biomedical studies, as release, which included in vivo dual-mode imaging the nanomaterial is often functionalized to achieve consisting in NIR fluorescence and MRI. improved stability or biocompatibility or it is loaded A step forward has been taken by Zuo et al. [251] with NPs or drugs, used for imaging or to deliver the with the delivery of dsDNA/siRNAs to Neuro-2a therapy, a careful analysis of the properties from cells, thanks to the development of innovative synthesis to the final vector is of crucial importance. manganese-based layered double hydroxide Recently, the community has introduced some nanoparticles, explored for anticancer drug/gene guidelines to establish universal practices to allow a delivery system and T1-weighted MRI for brain cancer better classification of solution-processed 2DMs, theranostics. based on specific parameters, such as thickness, size, In addition to imaging and drug delivery, Guan and surface chemistry [253]. However, et al. [248] developed a NIR-sensitive layered characterization of solution-processed 2DMs is supramolecular nanovehicle for a chemo-photo- currently very challenging because dispersions thermal synergistic therapeutic agent. The particular typically contain nanosheets with a wide distribution structure of Gd-LDH not only stabilized ICG, in size and thickness, in contrast to traditional enhancing the photothermal capacity, but also colloidal dispersions. Thus, a combination of different improved the recombination between electron and techniques (Table 5A, Table 5B), ranging from holes, generating more ROS under NIR irradiation. nanoscale to microscale resolution, must be used to characterize solution-processed 2DMs. Furthermore, 4. The importance of 2D material due to the different nature and dimensionality of characterization for theranostics solution-processed 2DMs, characterization methods Characterization of nanomaterials is of typically used for colloids may have limited validity, fundamental importance for the successful when applied to 2DMs. In this section, we provide an development of commercial applications [252], in overview of the most used techniques for particular in the context of human health and safety characterization of 2DMs used for theranostics (Table because the biological and toxicological properties of 5A, Table 5B). The following sections are dedicated to the nanomaterial are directly determined by its the different characterization methods (and refer to structure. Figure 3 for an overview).

Table 5A. Table summarizing the different methodologies for the characterization of GRMs.

Ref Material Characterisation Techniques

Acronym Microscopy Elemental Analysis/ Spectroscopy Zeta Potential/ Diffraction DLS/Stability/Others GRMs Luo et al. Chem CAD-SPIONs@GO TEM: GO height= XPS shows partial RS: confirm SPION growth Zeta: GO = -52 mV & Comm (2019) 0.5-1.1 nm & size= ~100 reduction.; on GO; FT-IR confirms CAD-SPIONs@GO = +201 mV;

GO nm. XRD confirmsGO functionalisation with DLS: GO = 127 nm & CAD; 1H NMR confirms CAD-SPIONs@GO = 175 nm;

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Ref Material Characterisation Techniques

Acronym Microscopy Elemental Analysis/ Spectroscopy Zeta Potential/ Diffraction DLS/Stability/Others GRMs functionalisation of GO TGA confirms with CAD functionalisation Diaz-Diestra et FA-rGO/ZnS:Mn QDs TEM: GO size =200-500 SAED confirms RS: confirms reduction of Zeta: increases from +5.5 mV to al. nm; SEM: ZnS:Mn NPs ZnS:Mn; XRD: peak at GO & functionalisation −18 mV with FA conjugation of

Nanomaterials well dispersed onto the 10° for GO, which with QD & ZnS; rGO-ZnS:Mn in PBS; Stability:

(2018) rGO surface of rGO. disappears for UV-Vis&FTIR: confirms Stable for 24 h, in cellular rGO/ZnS:Mn reduction & media supplemented with FBS functionalization. Usman et al. BIT TEM: confirms ICP‒ES & EDS: C & H UV-Vis: confirms drug TGA: confirms the composition PLoS ONE formation of the % increase from GO loading & release; of the hybrid (2018) hybrids; size= 200-500 to BIT. Gd & Au RS(532nm): I(D)/I(G) = 0.84 nm detected; XRD: peak for GO; =0.86 for GOGCA;

GO @ 10° for GO, = 0.94 for final BIT broadening & composite; FT-IR: confirms downshifts after functionalization. functionalization Usman et al. GAGPAu (or GOTs) HRTEM: AuNPs have XRD: peak @ 10° for RS(532nm): I(D)/I(G) = 0.84 TGA: confirms the

Molecules.(2018) size of 2 nm, size of the GO, broadening & for GO; 0.86 for GOGCA; composition of the hybrid NS (from pictures) ~500 downshifts after 0.94 for final composite; GO nm functionalization FT - IR : confirms functionalization.

Ko N.R. et al. G2, GQD–βCD, & GQD-comp, FE-SEM&FE-TEM: EDX: C, N, & O UV-Vis&FT-IR: confirms DLS: GQD- NH2 has size of 26 RSC Adv.(2017) DL-GQD GQD size= ~20-40 nm, elements surface functionalisation nm, GQD-comp has size of 222 GQD-comp height= 6-7 nm; TGA confirms the GQDs nm composition of the hybrid Bahreyni et al MUC1 aptamer-NAS-24 AFM: GO size= 195nm, FT-IR: confirms GO Int J Pharm. aptamer-GO, MUC1 slight increase after

(2017) GO aptamer-Cytochrome C complexing aptamer-GO Zhang C. et al. NGO-PEG–ICG/PTX AFM & TEM: NGO FT-IR: confirms Zeta: -29 mV; DLS: ~100 nm, no

RSC Adv. (2016) size= 70 nm & PEGylation; UV-vis: change over time in PBS & FBS NGO-PEG-ICG/PTX confirms conjugation with NGO size= 95 nm. Height= ICG & PTX. ~1-2 nm

Wang X. et al. J. AS1411–GQDs AFM: GQD size= ~30 UV-Vis: confirms Zeta: GQD = -21 mV Mater. Chem. B nm & height = ~1 nm. conjugation

(2015) GQDs Dong et al ACS f-GQDs TEM&AFM: GQD size= XPS: confirms GO FT-IR: confirms Zeta: GQD = -30 mV,

Appl Mater ~17 nm & height= ~1.3 functionalization GQDs-PEG = -15 mV, f-GQDs Interfaces. nm, f-GQDs size = 22 = -5 mV. nm, height = 1.7 nm; GQDs HRTEM: d-spacing of 2.5 Å Yang HW et al. Gd-NGO/Let-7g/EPI AFM: NGO-COOH & XPS: confirms UV-Vis: EPI adsorption Zeta: Gd-NGO= +48 mV &

Biomaterials. Gd-NGO size=~150 nm oxidation & confirmed Gd-NGO/Let/EPI= +33 mV;

(2014) NGO & heights= ~1.8 nm conjugation (22.8%). TGA: conjugation confirmed. &~3 nm. Wang C. et al GO-RGD-Chitosan AFM: RC–GO–DOX FT-IR& UV-Vis: confirm Zeta: GO= −33.6 mV, RC–GO– Colloids Surf B height= 2.3 nm, larger loading of DOX & RC; 1H DOX=+31.6 mV

Biointerfaces GO than height of GO NMR: confirms RGD (2014) modification of CS Nie L. et al. ACS GO-Cy5.5-Dox AFM: GO-PEG size = UV-Vis: confirms successful

Nano (2014) ~10 nm, conjugation of Cy5.5 & GO-PEG-Cy5.5-Dox DOX. GO size = ~30 nm & height= ~6 nm. Some S. et al Sci. GO-Cur, DGO-Cur & GQD-Cur SEM: confirms Cur XPS: C/O ratio= 2.2 FT-IR &UV-Vis: confirm Stability: GQDs were readily

Rep. (2014) loading. Cur NPs size = for GO; ~1 for DGO; Cur functionalisation of water-dispersible 120-150 nm; HRTEM: < 1 for GQDs GO, DGO & GQD GQD size= 3-6 nm; GQDs AFM: GO & DGO size= GO, GDO & 1-10 um.

He D. et al GO Capped Mesoporous Silica TEM: MS- NH2 size= XRD: confirms FT-IR&UV-Vis: confirm BA Zeta: MS-BA-GO = −29 mV. (Langmuir) ~100 nm, capping of uniform functionalisation &

(2014) GO MS-BA by GO is mesostructure of MS- PEGylation confirmed. NH2.

Chen M.L. et al Gr-HQDs TEM: Gr-PSS, Gr-HQD XPS: confirms UV-Vis: confirms DOX Zeta: Gr-PSS= −38 mV; (Bioconjugate to show morphology of reduction & loading & GO reduction & Gr-PSS-PAH-HQDs-Trf = +8.3 Chem.) (2013) NS & uniform functionalization; conjugation.; RS(633nm): mV; Stability: Gr-HQDs stable HQDs - distribution of HQD on EDX: confirms I(D)/I(G) = 2 for GO & 1.36 in water & biological solution Gr Gr conjugation, for GO-PSS. for 4 h at 37 °C Ma X. et al. J. Au@NGO TEM & FE-SEM: XRD: confirms Au in RS (488 nm): confirms GO Zeta: Au@NGO = -28 mV; DLS: Mater. Chem. B, Au@NGO size <100 nm the composite in the composite; FT-IR & Au@NGO= 98 nm in water &

(2013) GO UV-Vis: confirms 134 nm in DMEM. conjugation of AuNPs

Chen Y et al rGO/Au/Fe3O4 HRTEM: Fe3O4 NP XRD: rGO peak at RS: broad D & G peaks Zeta: GO= -40 mV; GO/Fe3O4

ACS & covered by thin layers ~18° visible, confirming the system shows positive zeta for Nano.(2013) rGO/BaTiO3/Fe3O4 of rGO (~2 nm height); presence of GO. pH <7 & negative zeta for pH rGO SEM: NP-rGO size= >7. 300-700 nm.

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Ref Material Characterisation Techniques

Acronym Microscopy Elemental Analysis/ Spectroscopy Zeta Potential/ Diffraction DLS/Stability/Others GRMs Zhang M. et al GO-DTPA-Gd AFM: GO size= 100−300 XPS: confirms DTPA FT-IR: confirms Zeta: GO-COOH= -47 mV,

ACS Appl. nm & height= ~1.0 nm. conjugation PEGylation; UV - Vis: GO - PEG = - 16 mV; Stability: no Mater. conjugation with DOX aggregation for 1 d in FBS & GO Interfaces, confirmed CCM (2013) Wang Y. et al MGMSPI SEM: MGMS size= RS(633nm): D, G & 2D b& TGA: confirms reduction of Gr Small (2013) ~200nm, smaller than confirm GO presence. when covered with MGMS GO; TEM: spherical I(D)/I(G)>1;

GO NPs evenly distributed FT-IR&UV-Vis: confirms on GO; HRTEM: Fe3O4 conjugation & reduction. NPs size= 4-15 nm. Wang C et al. J DOX–CMG–GFP–DNA TEM: CRGO size= FTIR: confirms DLS: CRGO= 126 nm, Mater Chem B ~150nm functionalisation; UV-Vis: DOX-CMG= 91 nm

Mater Biol Med. GO confirms DOX loading. (2013)

Shi S. et al SEM & AFM: size= 20- Zeta: RGO-PEG- NH2= -20 mV, Biomaterials 64Cu-NOTA-RGO-TRC105 80 nm NOTA-RGO-TRC105= -2 mV;

(2013) rGO DLS: RGO-PEG- NH2= 22 nm, NOTA-RGO-TRC105= 37 nm

Gao Y et al GO-SiO2 SEM & TEM: SiO2 size= FT-IR&UV-vis: confirm

Colloids & Surf. 70–80 nm, GO of conjugation of GO & SiO2 as

B Biointerfaces GO micron-size. well as DOX loading. (2013)

Hong H. et al. GO AFM: GO-PEG- NH2, Zeta: GO-PEG- NH2= -4.8 mV, ACS Nano NOTA-GO & NOTA-GO-TRC105= -0.1 mV;

(2012) GO NOTA-GO-TRC105 DLS: GO-PEG- NH2= 22 nm, have size= 0 - 50 nm. NOTA-GO-TRC105= 27 nm Kim H. et al GO-BPEI AFM: GO size= 500-600 FT-IR&UV-Vis: Zeta: GO=-30 mV, GO-BPEI=

Bioconjugate nm & height= 0.6-1.3 confirmation of BPEI +40 mV; Stability: stable in Chem. (2011) nm, BPEI-GO/pDNA functionalisation water, PBS & DMEM media for GO size= 300-400 nm & 1 hr height=16-18 nm. Sun X. et al nGO-PEG AFM: GO size= 10-300 FT-IR: confirms PEG

Nano Res. (2008) nm & height= ~1.0 nm, functionalisation; UV-Vis:

NGO NGO-PEG mostly <20 confirms conjugation. nm. Li P. et al. UCNP@NGO TEM: UCNPs size= ~55 FT-IR: to confirm DLS: NGO= ~100 nm &

Biomater. Sci. nm & NGO size= ~200 coordination of UCNP on UCNP@NGO= ~200 nm; TGA:

(2018) NGO nm NGO; 5 wt% of OA & 34wt% of UCNPs on NGO.

Bi H. et al. ACS GO/ZnFe2O4/UCNPs (GZUC) AFM: GO height= 1-2 ICP-MS: mass ratio UV-Vis: strong absorption Zeta: UCNPs = ~-12 mV, publications nm; TEM: UCNPs high GO:UCNPs:ZnFe2O4 peak of GO GZUC-PEG = ~-18 mV; DLS: (2018) monodispersity & size = ~1:2:2; XPS: Zn & Fe GZUC-PEG= ~400 nm.

GO <10 nm peaks; XRD: confirms ZnFe2O4 & hexagonal NaGdF4 in GZUC

Gulzar A et al. NGO-UCNP-Ce6 (NUC) AFM&TEM: NGO-PEG XRD: confirms UV-Vis: confirms Ce6 Dalton Trans size = ~100 nm, single UCNPs loading on UCNPs-NGO; (2018) NGO layer. FT-IR: confirms PEGylation

Zhang Y. et al. J. GO/Bi2Se3/PVP AFM&TEM: GO size = EDS: confirms C, Bi & FT-IR: confirms GO & Zeta: GO= -18 mV; DLS:

Mater. Chem. B. 100–500 nm & height = Se in GO/Bi2Se3/PVP; functionalisation GO/Bi2Se3/PVP= ~ 149 nm

(2017) GO ~1 nm; HRTEM: NPs XRD: GO peak @ 11.4° size = ~3-9 nm. Gao S et al CPGA TEM: CPGA size= ~230 UV-Vis confirms composite Zeta: CPGA= -25 mV; DLS: Biomaterials nm, NP loading shown; formation ~230 nm; Stability: 1 d storage

(2016) GO AFM: CPGA height= (UV-vis-NIR). ~15 nm. Kalluru P et al. GO-PEG-folate-mediated NmPDT AFM&TEM: ~100 nm RS: D & G peak visible; DLS: ~100 nm

Biomaterials size FT-IR: confirms composite;

(2016) NGO UV-Vis: confirms functionalization of GO Lin LS et al. PEG-rGO-GSPs TEM & AFM: GO size= UV-Vis: confirms reduction DLS: 3 GO-GSPs with size: 60

Nanoscale. ~150 nm, GNPs= ~14 of GO; RS: broad D & G nm, 90 nm & 130 nm; Stability:

(2016) rGO nm peak visible stable in water, PBS, CCM & FBS for 3 d Luo S et al. ACS NGO-808 AFM&TEM: NGO size= FT-IR: confirms

Appl Mater 100-500 nm & height= 1 functionalization; 1H NMR:

Interfaces. NGO nm, NGO-808 size = 20 confirms PEG (2016) 40 nm & height= ~3 nm. functionalization Hu D et al ICG-PDA-rGO AFM: GO size <1 µm, XPS: shows some GO UV-Vis: confirms Theranostics. height= ~0.86 nm, reduction after conjugation with ICG

(2016) GO ICG-PDA-rGO no size functionalisation. change, height>2 nm. Huang G et al IO/GO-COOH AFM&TEM: GO-COOH FT-IR: to show COOH Stability: no aggregation for Nanoscale = 300-600 nm size & functionalisation. 30d (2015) height= ~2 nm,

GO IO-13/GO-COOH similar size & height= ~25 nm.

G Kim YK et al. O PEG-Au@GON NPs AFM&TEM: GON size= Elemental analysis: FT-IR: confirms conjugation Zeta: Au@GON NPs= −58 mV,

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Ref Material Characterisation Techniques

Acronym Microscopy Elemental Analysis/ Spectroscopy Zeta Potential/ Diffraction DLS/Stability/Others GRMs Small. (2015) 10 -15 nm & height= 1.2 C/O ratio=0.40; XPS: of Au; RS: I(D)/I(G) of GON=−47 mV; Stability: nm; TEM: spherical GO & Au peaks GON= 0.79 & Au@GON PEGylated composite showed shape of Au@GON observed. =0.83. better stability after 20min size= ~60 nm, NPs with storage (UV-Vis) 2–3 nm thick GON shell. Rong P. et al. GO-PEG-CysCOOH AFM: GO size = ~200 UV-Vis: Confirms Stability: Good stability in

RSC Adv. (2015) nm, GO-PEG size <~50 successful water, PBS, cell medium, FBS nm & height= ~1.5 nm, functionalization; GO GO-PEG-CySCOOH height = ~2 nm. Yan X et al. GO-PEG-DVDMS AFM: GO-PEG size= ~ UV-Vis: confirms

Nanoscale. 14 nm & height= 1.3 functionalization with (2015) nm. GO-PEG-DVDMS DVDMS GO size= ~ 20.5 nm & height= 1.5 nm. Zhang H et al. GO/BaGdF5/PEG TEM&AFM: GO size= XRD: confirms FT-IR: confirms Zeta: GO= −20 mV; Stability: Biomaterials. 200 nm & height= BaGdF5 formation; functionalization; UV-Vis: good stability in water, saline,

(2015) GO ~1.0 nm, smaller size of GO peak @ 11.3° confirms reduction of GO PBS, RPMI-1640 & FBS; TGA: GO/BaGdF5/PEG.∼ disappears BaGdF5= ~42 wt%

Gollavelli G. et MFG AFM: size= 40 nm FT-IR: confirms Stability: MFG–SiNc4 stable in al Biomaterials functionalization water & in biological media (2014) MGF ∼

Viraka Nellore ICG-FeCl3 @GO TEM: NP size= ~40 nm, RS: confirms GO TGA: magnetic NPs= 30 wt% & et al. Faraday confirms conjugation. GO= 70 wt %. GO Discuss. (2014) Nergiz S.Z. et al GO & GOAuNS AFM: GO size= 0.5 RS (785 nm): confirms Gr; Zeta: GO= -35 mV, GOAu= -25

ACS Appl. μm & height= 1 nm. No no large changes after Au mV; stable @ ~-20 mV in 10% Mater. Interfaces change after Au ∼NPs NPs formation. FBS; Stability: excellent GO (2014) formation. stability in 10% FBS over a course of 10 d. Wang S. et al GO-ION-PEG TEM: ION uniformly ICP-AES: Fe =55.6 RS: confirms GO & NPs; DLS: ~166 nm; Stability: good Biomaterials grew; AFM: NS size < wt% in GO-IONP. UV-Vis: confirms reduction stability in water &

(2014) GO 200 nm & height= 3-10 of GO; FT-IR: confirms physiological solutions for 72 h nm. fucntionalization with PEG Rong P et al. GO-PEG-HPPH AFM: GO-PEG size< 50 UV-Vis: confirms loading of Stability: good stbaility in ADV nm & height= ~1.5 nm; HPPH water, PBS, cell medium &

Theranostics GO GO-PEG-HPPH serum (2014) height= ~2 nm.

Cho Y et al. GO–HA–Ce6 SEM: 500-700nm size DLS: GO complex= ~441 nm Chem Commun (one picture) GO (Camb) (2013) Shi X et al. GO-IONP-Au-PEG AFM: GO & GO–IONP Zeta: GO–IONP= −35 mV,

Biomaterials. size= ~200-600 nm. GO-IONP-Au-PEG= −5.6 mV; (2013) Stability: GO-IONP-Au-PEG GO stable in water, saline & serum solutions.

Wang Y.W. et al. ICG-Rgo-FA AFM: GO size = ~200 J. Mater. Chem. nm & height = ~1 nm B(2013) rGO

Sheng Z. et al BSA/nano-rGO AFM: height=4 nm; size XPS: confirms UV–Vis: confirms BSA Stability: BSA/nano-rGO

Biomaterials rGO = ~70 nm reduction absorption stable in buffers & other - (2013) biological solutions for one

nano month's storage Yang K. et al. RGO–IONP–PEG TEM: IONPs size= 8–10 XPS: confirms FT-IR: confirms PEG Stability: RGO–IONP–PEG Adv. Mat. (2012) nm, even distribution reduction after IONPs functionalization; NMR: ~ stable in water, saline & serum

on RGO; AFM&DLS: growth; ICP-MS: 20% of carboxyl groups solutions RGO–ION size> 200 RGO:IONP weight conjugated with PEG rGO nm; RGO–IONP–PEG ratio= 1:1.99; XRD: size= ~50 nm confirms IONP formation Hu S.H. et al rGO-QD TEM: well‐ordered QDs DLS: Three batches with

Adv. Mat. (2012) (size= 3.6 nm) array different size: ~2.5 μm; ~260

rGO separated by gaps of 1.7 nm; ~38 nm; Stability: QD‐ nm. rGO highly stable.

Yang K. et al NGS-PEG AFM: size= 10−50 nm & FT-IR: confirms Stability: good stability of Nano Lett. height= ~1 nm functionalization functionalized GO (2010) NGS Karimi RGO-PDA-BSA-DTPA-Mn(II)/MTX AFM: GO NS height = FT-IR: confirms Shervedani R et 3.9 nm; RGO-PDA-BSA functionalization; After al. Biosens height= 17 nm. Smooth functionalization of GO Bioelectron. surface/good with PDA, reduction is (2018) RGO distribution of confirmed. Mn(II)/MTX (height= ~0.9 nm).

Chang X. et al. GO/MnWO4/PEG TEM: GO size = ~130 XPS: Mn, W, C, O, & FT-IR & UV-Vis-NIR: Zeta: GO = ~-42 mV,

Carbon (2018) nm, MnWO4 NPs size= N; XRD: confirms confirms GO & conjuataion GO/MnWO4/PEG = ~-26 mV; ~12 nm; AFM: MnWO4 TGA: 59.6 wt % of MnWO4 & GO GO/MnWO4/PEG 13.0 wt% of GO in height=~16 nm, GO GO/MnWO4/PEG.

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Ref Material Characterisation Techniques

Acronym Microscopy Elemental Analysis/ Spectroscopy Zeta Potential/ Diffraction DLS/Stability/Others GRMs height= ~3.4 nm; SEM: confirms conjugated morphology

Gulzar A et al. UCNPs-DPA-NGO-PEG-BPEI-DOX AFM: height= 30 nm; FT-IR: confirms PEG Dalton Trans TEM: size= ~100 nm functionalization (2018) NGO Wu C et al. Acta GO/AuNS-PEG & TEM: GO size = ~ 2 μm, UV-vis: confirms Au NPs Zeta: GO/AuNS-PEG/Ce6= – Biomater. (2017) GO/AuNS-PEG/Ce6 Au NP coating (~40 nm on GO 38 mV, GO/AuNS-PEG= -20

in size) mV; Stability: GO/AuNS-PEG AFM: stable; TGA: content of PEG is GO GO/AuNS-PEG/Ce6 ~40% height = ~18 nm & size= ~400 nm. Battogtokh G. et PheoA + GO:FA-BSA-c-PheoA NC UV-Vis: confirms Zeta: GO NP= -24 mV; DLS: al. J. Control. conjugation; GO = ~249 nm,

Resease(2016) GO PheoA+GO:FA-BSA-c-PheoA NC (0.5:1 w/w)= 182 nm Justin R et al. MGQDs-LH TEM: Iron oxide coating RS (514.5 nm): very small & DLS: ~61 nm

Carbon (2016) on Gr surface; HRTEM: broad G peak, intense & several layers rGO, very broad D peak; FT-IR: wrinkles; AFM: flakes confirms Iron oxide GQDs diameter= 45nm & formation height= 2.3 nm. Chen Y.W. et al. anti‐EGFR‐PEG‐rGO@CPSS‐Au‐ TEM: GO/silica NS EDX: Si, O, & C in UV-Vis & FT-IR: confirms

Small (2016) R6G height = ~44 nm. SEM: GO/silica. Au in conjugation with Au NPs. confirms morphology rGO@CPSS-6-Au; rGO of rGO@CPSS-x, size = XRD: confirms ~160 nm composite

Chen H et al GO-Au@PANI/DOX AFM: GO-PVP height= RS (785 nm): GO peaks not Stability: GO-Au@PANI very Theranostics. ~1.3 nm; TEM: unevent visible due to polymer stable in water, PBS & GO (2016) deposition on GO RPMI-1640 Shi J et al. GO@Gd-PEG-FA/DOX TEM: UV-Vis&FT-IR: both Zeta: GO@Gd-PEG-FA/DOX Pharm Res. GO@Gd-PEG-FA/DOX confirm functionalization =−6 mV; DLS: GO-Au@PANI=

(2016) size is much smaller with PEG & FA 120 nm; Stability: than GO@Gd. GO@Gd-PEG-FA very stable in GO water & physiological solutions; TGA: PEG in GO@Gd= 15.2 wt%

Miao W et al. J ICG/HArGO & ICG/rGO TEM: shows four flakes Zeta: rGO= -30 mV; all Control Release. with size ~ 100nm. hybrids= -50-60 mV; DLS: ~100 (2015) rGO nm for all materials. Yan X et al. GO-PEG-DVDMS AFM: GO-PEG size <50 UV-Vis: shows a new peak

Biomaterials. nm & height= 1.5 nm; for GO-PEG-DVDMS; (2015) GO-PEG-DVDMS GO height= 2 nm ∼& the size x2 larger. ∼ Chen H. et al. DOX–rGO–Fe2O3@Au NPs TEM: UV-Vis: to confirm Zeta: rGO-COOH = -29.5 mV, RCS Adv. (2015) NH2-PEG-Fe2O3@Au conjugation with rGO-Fe2O3@Au NPs = -21.1

NPs = ~20 nm & NH2-PEG-Fe2O3@AuNP; mV; DLS: rGO-COOH= 616

rGO rGO-COOH = ~1 µm. RS: confirms GO nm, rGO-Fe2O3@Au NPs= 612 Conjugation nm; Stability: Stable in DMEM morphology confirmed. + 10% (v/v) FBS for 5 h Khatun Z. et al. GDH TEM: Gr size= ~5-10 FT-IR: confirms conjugation Stability: stable in PBS, pH5 &

Nanoscale Gr nm, GDH nanogel size= 10% FBS solution for 7 d. (2015) ~100 nm

Yang Y et al. J. GO/MnFe2O4/DOX TEM: GO size= XRD: MnFe2O4 Stability: The hybrids can be

Biomater. 50-500 nm & height= deposition confirmed. well dispersed in water, Appl.(2015) 0.8–1.1 nm; physiological saline, & FBS GO GO/MnFe2O4 size= 70– 310 nm. Bian X. et al Sci. NGsAu nanocrystal TEM: core-shell size= 65 RS (633 nm): I(D) ≈ I(G), Zeta: ~0 mV; DLS: confirms

Rep. (2014) nm. Few-layers Gr as indicating high defect particles size; Stability: Gr uniform coating on the concentration. functionalization increases whole particle. stability. Feng L et al. GO-PEG-DA AFM: size of 50-100 UV-Vis: confirms Zeta: GO= -40 mV; Adv Healthc nm. functionalization NGO-PEG-DA= -35 mV; DLS:

Mater. (2014) confirms stability; Stability: all

NGO stable in physiological solutions; TGA: PEG content= ~ 25.3% Shi J. et al GO-Ag TEM: confirms presence XRD: GO peak @ UV–Vis: confirms Ag NPs; Zeta: GO-Ag-DOX-NGR= −30

Biomaterials of Ag NPs with 10.9°, disappeared FT-IR: decrease in O–H mV (2014) diameters of 5–15 nm completely in GO-Ag. peak after activation; GO confirmed DOX conjugation Jin Y. et al. Au@PLA-(PAH/GO)n TEM: GNP size = ~2.6 - UV-Vis & FT-IR: confirm

Biomaterials 2.8 nm, TEM & SEM: conjugation (2013) microcapsule GO morphology, size = ~1 µm

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Ref Material Characterisation Techniques

Acronym Microscopy Elemental Analysis/ Spectroscopy Zeta Potential/ Diffraction DLS/Stability/Others GRMs Wang Y. et al GSPI AFM: GO size= ~50–250 XRD: Gr is coated RS (633 nm): D peak Zeta: GO = −52 mV; GO-PEG = J.Am.Chem.Soc nm; TEM: confirms with a hexagonal indicates defective Gr; −38 mV; stability: GSPI retains

(2013) Gr mesopores with size of array of pores with FT-IR: indicates GO water dispersion. 50–150 nm; HRTEM: ordered structure. reduction & confirms pore depth= ~25 nm. functionalization.

Wang Y. et al GO-UCNPs--ZnPc GO-PEG height= 1.2 FT-IR: confirms PEG Biomaterials nm, size = 100-250 nm functionalization GO (2013) ∼ Miao W. et al Ce6/Dox/pGO AFM: pGO NS height= FT-IR: confirms Zeta: ~−41 mV for all Biomaterials ~1 nm & functionalization in pGO complexes.

(2013) GO Ce6/Dox/pGO size = Note: inconsistency between ~148nm AFM & DLS size data of pGO.

Qin H et al. (GO-dye: GO–RhB, GO–Cy5, & GO– Stability: All stable in Small (2015) Cy7)GO–Abs/Cy7 physiological solutions (but not GO NGO) Hatamie S et al Gr/cobalt nanocomposites TEM: confirms sheet XPS: O/C ratio=0.9 FT-IR&UV-vis: indicates Zeta: ~ -18 mV (in PBS) Colloids Surf B decorated by the NPs. for GO, 0.18 for GO reduction after hybrid Biointerfaces. composites; XRD: formation; RS(532 nm):

(2016) GO indicates GO confirms reduction reduction after hybrid (although not complete) formation. Chen L et al. 131I-RGO-PEG AFM: RGO-PEG Stability: GO-PEG is stable in

Biomaterials size= 40-60 nm & water, saline, serum; (2015) heights= 3–4 nm. 131I-RGO-PEG is stable in 9% RGO ∼ NaCl, FBS, PBS, & RPMI-1640 for 7 d

Table 5B. Table summarizing the different methodologies for the characterization of new 2DMs.

Ref Material Characterisation Techniques 2DMs Acronym Microscopy Elemental Analysis/ Diffraction Spectroscopy Zeta Potential/ DLS/Stability/Others

Yang G. et al. TEM (HAADF-STEM): WS2 Elemental mapping: Weight ratio UV-vis: confirms Stability: WS2-IO@MO-PEG in water,

2 PEG -

Small (2018) IO/S size= ~50 nm. of W:Si:Fe:Mn = 1:5.02:4.02:7.6 composite formation PBS, FBS, CCM for 24hr shows good - 2 WS stability (DLS) WS

@MO - Chao Y. et al. 2 TEM (DLS): WS2 -PEG size= UV–Vis: WS2- PEG do not Stability: PEGylated nanoflakes

2

Small (2016) WS 80–150 nm show any feature exhibit high stability in various - WS PEG physiological solutions Re

188

Yong Y. et al. TEM: WS2 size= 20–100 nm; EDS: W, S, C; XRD: confirm FT-IR: confirms Stability: BSA–WS2 shows good

Nanoscale 2 AFM: WS2 height= ~ 1.6 nm, hexagonal structure of WS2 functionalization with stability in PBS

2

(2014) WS BSA-WS2 height= 4–5 nm. BSA; UV-Vis: sharp peak @ -

WS ~610 nm; RS: peaks are

BSA broader compared to bulk WS2.

Cheng L. et al. AFM: WS2 height= ~ 1.1 nm. EDS: W & S; XRD: confirms UV-Vis: WS2-PEG NS Stability: WS2 PEG exhibits excellent

2

Adv. Mater. PEG WS2- PEG height = 1.6 nm.; hexagonal structure of WS2. show typical features stability in various physiological - 2

(2013) WS TEM: WS2 -PEG size= 50-100 observed for 2H-WS2 solutions

WS nm ∼ phase.

Liu J. et al. AFM(DLS): MoS2 & MoS2-HA- EDS&XPS: confirms even FT-IR & UV–Vis: confirm Stability: MoS2-HA- NH2 exhibited J. Colloid NH2 sheet-like morphology. distribution of Gd on the surface functionalization with HA; enhanced stability in water, PBS & cell

DTPA

2 Interface Sci. - MoS2 NS height= 1-2 nm. of the MoS2. UV - Vis: different from 2H medium; TGA: HA-SS- NH2= 67.4 Gd - HA

(2019) MoS MoS2-HA height= 3-4 nm. MoS2 -no exciton peaks wt% in MoS2-HA- NH2. - 2 MoS2= ~100 nm, MoS∼ 2-HA- visible. ∼ ∼

MoS NH2 size= ~200 nm

Jing X. et al. TEM: Mn-Fe3O4@MoS2 has a XPS: confirms MoS2, & Fe3O4; RS(514 nm): confirms Zeta : Mn-doped Fe3O4= –26 mV; 2 Bioconjugate petal shape, a rough & folded confirms Mn doping; XRD: heterojunctions between DLS: all hybrids = 200–1000 nm.

2 Chemistry surface structure; DF-SEM: confirms magnetite Fe3O4 & MoS2 & Mn-doped Fe3O4, Stability: all stable for 5 d in DI water. @MoS doped 4 -

(2018) MoS confirms Mn-doped Fe3O4 is MnFe2O4. Diffraction peaks of MoS2 peaks. FT-IR& The composites are stable in DI water O 3

Mn located at the core of the pristine 2H-MoS2 of Mn-doped UV-vis: confirm & PBS for 5 d. nanoflower Fe flower-like MoS2 NS. Fe3O4. functionalization.

Maji S. et al. SEM: 1 NS of size= ~80 nm; EDX: confirms the formation of RS: MoS2 peaks visible in

2 2 ACS Appl. TEM: thin layer of MoS2 with core@shell AuNBPs@MoS2. AuNBPs@MoS2; UV-Vis:

Mater. MoS MoS 1.5-2 nm observed on AuNP no excitons peak observed.

Interfaces(2018) AuNBPs@ surface.

2 2 2

Dong X. et al. AFM: MoS height= ~0.8 nm, RS (514 nm): a slight shift Zeta: MoS = ~-20 mV, MoS -PEI-HA= ACS Appl. MoS2-PEI-HA height= 5-7 nm; compared to the bare -18 mV; Stability: improved stability HA

- Mater. 2 TEM: MoS2 size= ~30-50 nm. MoS2; FT-IR: confirms after HA modification in water &

Interfaces PEI conjugation of PEI & HA; other physiological solution. - 2 (2018) MoS UV-Vis: exciton peaks of

MoS MoS2 visible in conjugated material

Chen L. et al. MoS2 MoS2 & MoS2-Gd-BSA - XPS: confirms MoS2. RS(633 nm): typical MoS2 Zeta: MoS2= -30 mV, MoS2-Gd-BSA = BS -

ACS Appl. 2 composed of several lamellar peaks; FT-IR: confirms -26.4 mV; DLS: MoS2= 205 nm,

Gd A Mater. - structures, forming cluster. functionalisation. MoS2-Gd-BSA= 297 nm; Stability: 2 MoS Interfaces MoS2-Gd-BSA in PBS & CCM; TGA:

(2017) MoS confirms functionalisation.

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Ref Material Characterisation Techniques 2DMs Acronym Microscopy Elemental Analysis/ Diffraction Spectroscopy Zeta Potential/ DLS/Stability/Others -

Liu B. et al. 4 SEM&TEM: composite size EDS: Mo, S, N, Fe & O. ICP-MS: FT-IR&UV-Vis: confirm Zeta: MoS2= -25 mV, MoS2-PEI= 27 O 3

Adv. Sci.(2017) 2 ~80 - 180 nm, no morphology 5.1 wt% of Pt; XRD: shows functionalization with ICG mV; Stability: no aggregation for 2 d ;

@Fe change with functionalization. 2H-MoS2 phase TGA: 27.8wt% of PEI 2 MoS HRTEM&HAADF/ anoflowers ICG/Pt(IV)N MoS STEM-EDS: confirm Fe3O4.

Xu J. et al. SEM&TEM: bulk MoS2 flower EDS&XPS: confirm successful FT-IR&UV-Vis: confirms Zeta: MoS2= -25 mV, MUCS = +5 mV;

Small (2017) 2 2 structure & NS size=~500 nm, conjugation; XRD: confirms successful Stability: MUCS in DMEM for 1d spherical UCS size= 48 nm & successful conjugation functionalisation & ( UV - V is ) MoS MoS composite structure with UCS conjugation particles on NS are shown.

Zhao J. et al. TEM: MoS2 size= ~50 nm, XPS&EDS: confirms MoS2, EDS: FT-IR: confirms successful Stability: MoS2-PVP in water, saline,

2 PVP

Oncotarget - MoS2-PVP size= 21 nm, Mo, S, C & O; XRD: confirms functionalisation. CCM, & FBS showed good stability 2

(2017) MoS height= ~ 1 nm, for higher PVP crystalline structure of MoS2 for 3 d

MoS content size = ~15 nm

Li X. et al. Int. J. 2 TEM&FESEM: crumpled sheet UV-Vis: SP-MoS2 peak ~ Stability: SP-MoS2 stable in water,

2 MoS

Nanomed - structure of SP-MoS2 size= ~50 ~300nm saline, & RPMI - 1640 for 2d ( DLS ) MoS

(2016) SP nm

Yu J. et al. 3 SEM&TEM&AFM: MoS2 size= EDX&XPS: show possible surface FT-IR: confirms Zeta: MSIO= -22 mV, MoS2= -23 mV;

2

4 Theranostics /Fe ~100 nm, & height= 8-12 nm oxidation of MoS2 conjugation ; RS: MoS 2 DLS: MSIOs = ~190 nm in water& PBS 2 O

(2015) MoS peaks visible for 2 d; Stability: MSIO in DMEM,

MoS FBS. S

Wang S. et al. 2 FESEM&HRTEM: crumpled & EDS: Mo, Bi, S, & O; XPS: confirms FT-IR: confrims PEG TGA: 20.7 wt% of PEG (100-350°C)

2

Adv. /Bi defective MBP size= ~300 nm, composite formation; XRD: functionalisation 3 2

Mater.(2015) MoS MoS2 crystal lattice= 0.26 nm confirms high purity of the

MoS composite.

2 Wang S. et al. FESEM&TEM: MoS2 NS size= EDS: Mo & S for MoS2, additional FT-IR: confirms Stability: No change in DLS in saline Biomaterials ~50 nm & height= 0.25 nm, C & O for PEGylated composites; PEGylation after 24hr storage

(2015) 2 MD300-PEG size= ~297 nm & XPS: confirm PEGylated

MoS height= 0.42 nm. composite; SAED: confirms crystallinity; XRD: confirms

PEGylated MoS PEGylated PEGylated composites.

Liu T. et al. HRTEM: MoS2 NS size= 50-200 EDS: Mo, S, Fe, & O; XRD: Zeta: MoS2= -12 mV, MoS2-IO-(d)PEG

(d)P 2 -

ACS Nano nm, confirms MoS2 hexagonal confirms MoS2 NS. = +2 mV; DLS: ~100 nm size for all IO

(2015) - structure. composites; stability: good stability EG 2 MoS for MoS2-IO; TGA: confirms

MoS PEGylation

P 2 - Liu T. et al. AFM: MoS2 size= ~100 nm & UV-Vis: confirms DLS: ~200 nm; Stability: MoS2 & 2 Nanoscale height= ~1 nm, MoS2-PEG functionalisation MoS 2 - PEG in water & PBS. MoS 2 EG MoS

(2014) MoS height= ~2 - 10 nm. unstable in PBS, but the others stable.

Li Z. et al. ACS TEM & AFM: p-BPNSs size XPS: confirms no oxidation in RS (633 nm): confirms Zeta: p-BPNSs= −40 mV; DLS: Appl. Mater. <200 nm & height= ~1.3 nm; p-BPNS BPNS; UV-Vis: confirms p-BPNSs= ~200 nm; Stability: stable in Interfaces HRTEM: 0.22 nm lattice fringe; no degradation of p-BPNS; water, PBS, RPMI & FBS. BPNSs - BP

(2019) p TEM: morphology change of FT-IR: confirms - BPNS after 7 d, no change of conjugation RP p-BPNS.

Huang H. et al. TEM & AFM: BP size= ~500 EDS: BP/Bi2O3 = P 36.8%, O Zeta: BP is -27 mV, BP/Bi2O3 is +34 3 O

2 2 3

Biomaterials nm, Bi O NPs size = 5 nm, 56.5%, & Bi 6.7; XPS: P ~35.51%, O mV.; Stability: BP unstable but

(2018) BP BP/Bi2O3, size = 300 nm & ~54.99% &~Bi 9.5%.; XRD: BP/Bi2O3 stable for 8 d (optical, height = 25 nm; HRTEM: 0.21 confirms Bi2O3 absoprtion spectroscopy) BP/Bi nm d-spacing of BP Qiu M. et al. TEM & AFM: BPNSs size = XPS: confirms BP RS (514 nm): confirms BP Zeta: BPNS= -28 mV & PEG-BPNSs=

Proc Natl Acad ~100-200 nm & height = 2.6 in composites -17 mV; DLS: BPNSs =156 nm & PEG- BP Sc (2018) gel nm; HRTEM: d-spacing of BPNSs = 160 nm

BP@Hydro BPNSs 0.34 & 0.42 nm Yang X. et al. TEM &AFM: BP size= 90 nm, EDX: confirms mostly P & little RS (633 nm): confirms BP; Zeta: BP NSs = ~-17 mV, ACS Appl. height= 14.3 nm, BP-PEG size= oxidation; XRD: confirms BP UV-Vis-NIR: confirms BP@PEG/Ce6 NSs = ~-8 mV; DLS: BP

Mater. 100 nm, height= 15 nm. crystallinity loading of Ce6 NSs & BP@PEG NSs= ~ 90 nm, BP Interfaces NSs BP@PEG/Ce6 NSs = 158 nm; stability: (2018) BP & composites in water & in PBS BP@PEG/Ce6 confirmed for 24 hr; Yang B. et al. TEM &AFM: BP siz=e ~500 XPS: shows very small amount of RS & FT-IR: confirms Stability: stability of BP in SBF after 4

2 5

Adv. nm, height <10 nm, HRTEM: BP compared to P O , increased O biomineralization d shows flocculation & BG -

Mater.(2018) BP 0.32 nm d-spacing; TEM: & Ca% after biomineralization; biomineralisation BP

scaffold morphology change after SAED: confirms BP crystal; XRD: biomineralizaion confirms SiO2 (cristobalite)

Chen W. et al. TEM & AFM: BP size= ~200 XPS: confirms BP with small UV-Vis & FT-IR: confirm DLS: BP = 281 nm; Stability: stable in

Adv. Mater. BP BP nm, height= ~5.5 nm. oxidation drug loading water for 24 hr (2017) F

Tao W. et al. - TEM&AFM: BP size =~120 nm XPS: P, C, & O in BP-PEG; FT-IR: confirms Zeta: BP = -13 mV, BP-PEG-FA = -17

Adv. & height =~1–2 nm, BP-PEG STEM-EDS: C, O, N & P in functionalisation; RS: mV; Stability: BP-PEG stable in PBS & BP PEG NSs Mater.(2017) - size = ~100 nm & height = ~2-3 BP-PEG-FA; XRD: confirms confirms BP in composites FBS (DLS= ~130 nm) for 7 d A/Cy7 A/Cy7

BP nm orthorhombic BP in BP-PEG

Yang G. et al. TEM: AuNPs size= 26 nm & UV-Vis: confirms Stability: stability of BP-Au & Biomaterials BP–Au size= 484 nm. conjugation with Au; RS BP-Au-/PEG in PBS solution BP

Science (2017) Au NSs (514.5 nm): confirms BP in -

BP composite

2

Xiang H. et al. TEM: Nb2C size= 100-300 nm, XPS: confirms Nb (8.41%), C FT-IR & UV-Vis: confirms Zeta: Nb2C = −39 mV, ACS Nano morphology change observed (25.96%), Si (25.96%), & O (52.68%) AIPH functionalisation AIPH@Nb2C@mSiO2 = -10 mV; DLS: (2019) with functionalisation in Nb2C@mSiO2 NPs.; XRD: Nb2C = 219 nm, AIPH@Nb2C@ Mxene C@mSiO 2 AIPH@Nb confirms amorphous SiO2 mSiO2 = 308 nm.

3 2 @ A M xe C ne 3 2 3 2 3 2 3 2 Tang W. et al. Ti TEM&SEM&AFM: Ti C size = EDS: confirms Au on Ti C ; XRD: UV-Vis-NIR: confirms Zeta: Ti C = -24 mV, Ti C -PAH-Au =

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Ref Material Characterisation Techniques 2DMs Acronym Microscopy Elemental Analysis/ Diffraction Spectroscopy Zeta Potential/ DLS/Stability/Others

ACS Nano ~200 nm & height = ~2 nm; confirms Ti3C2 Mxene conjugation with Au. -20 mv; DLS: Bi3C2= ~100 nm, (2019) TEM: Au shell coating of ~30 Bi3C2@Au= 150 nm; Stability: nm height Ti3C2@Au-PEG in water, PBS, FBS & RPMI confirmed.

Liu Z. et al. TEM: Ta4C3 NS size = ~50-200 EDS: confirms Ta, C, O, & Fe in FT-IR: confirms Zeta: Ta4C3 =-24 mV &

Theranostics SPs nm, Ta4C3-IONPs size = ~50 - Ta4C3-IONPs composite; XPS: conjugation Ta4C3-IONP-SPs = -49 mV; Stability: - (2018) 200 nm; HRTEM: d-spacing confirms conjugation with IONPs; Improved stability with surface Ta4C3= 0.26 nm, IONP = 0.30 XRD: confirms Fe3O4 modification in water, DMEM, PBS, IONP - 3 Mxene nm saline, & SBF over 3 d; DLS: C 4 Ta4C3-IONP-SP = 255 nm, Ta Ta4C3-IONP = 190 nm.

Han X. et al. TEM: Nb2C NS size= several EDS&XPS: confirms conjugation; DLS: Nb2C = 91 nm, Theranostics hundreds nm; SEM&TEM: SAED&XRD: confirms retained CTAC@Nb2C-MSN-PEG-RGD = ~220 MSN (2018) - CTAC@Nb2C-MSN composite high crystallinity nm; Stability: stable in water, DMEM, Mxene C 2

CTAC@Nb of mesoporous morphology saline, SBF, & PBS (DLS)

Li Z. et al. TEM&SEM: Ti3C2 NS size= EDS&XPS: confirms conjugation FT-IR: confirms Zeta: Ti3C2 = -20 mV,

Adv. 50-100 nm with mesoporous functionalization. Ti3C2@mMSNs-RGD = 2 mV; DLS:

Mater.(2018) s morphology Ti3C2 =92 nm, Ti3C2@mMSNs-RGD = @mMSN 2

Mxene 2

C 152.9 nm; BET: surface area 772 m /g, 3

Ti pore size 3.1 nm

Han X. et al. SEM&TEM&AGM: Ti3C2 NS EDS & EELS: confirms complete Zeta: Ti3C2 = -52 mV, Dox@Ti3C2-SP = SP -

Adv. Healthc. 2 size = ~120 nm & height =~0.9 removal of Al -29 mV; DLS: Ti3C2 = 122 nm, Ti3C2-SP C

Mater.(2018) 3 nm; HRTEM: comfirms = 164 nm. Mxene Ti cyrstallinity

Liu et al. AM&I TEM&AFM: Ti3C2 size = ~100 XPS: confirms Ti-C & Ti-O in UV-Vis: confirms Zeta: Ti3C2 = -23 mV, 3+ (2017) nm & height=1-2 nm. Ti3C2; ICP: ratio of Al:Ti = 0.62:3; conjugation of Al Ti3C2@DOX@HA = -21 mV; DLS:

XRD: confirms Ti3C2 & Ti3C2 = 104 nm, Ti3C2@DOX@HA = A

@DOX@H conjugation 178 nm; Stability: Ti3C2 in water, PBS 2 Mxene C

3 compared with surface modified

Ti Ti3C2

Lin H. et al. SEM&TEM&AFM: Nb2C NSs EDS&EELS&XPS: confirms no Al RS: confirms elimination of Zeta: Nb2c = -50 mV; TGA: PVP JACS (2017) size= 150 nm & height= & presence of Ni, C & O; XPS: Al layer; FT-IR & loading 20 wt% PVP -

C ~0.3−0.8 nm confirms oxidation; SAED: UV-Vis-NIR: confirms 2

Mxene ∼ confirms hexagonal symmetry; functionalisation with Nb XRD: Nb2AlC MAX phase PVP;

S

Dai C. et al. - SEM&TEM: Ta4C3 NS size= EDS&XPS: confirms Ta, Mn & O in 3

C P

ACS Nano 4 ~50 - 100 nm MnOx/Ta3C3; XRD: confirms Mxene Ta (2017) MnOx/ removal of Al & 2D crystallinity

on the same sample in order to achieve a realistic distribution of the size and thickness of the flakes. However, in the case of solution-processed 2DMs, adsorption of solvent molecules or chemical modification of the surface of 2DM can lead to overestimation of the thickness (number of layers) of the nanosheets [256]. Thus, AFM can be used only as a qualitative method for thickness identification. TEM, on the other hand, can be used to directly count the number of layers by analyzing the flake edges and characterize the structure at sub-nanometer resolution in order to identify possible defects [257]. Low-resolution TEM can be used to look at the shape Figure 3. Analysis of the literature based on methods of material characterization for of the flakes and also to qualitatively identify the GRMs and other 2DMs. thickness by looking at the contrast. However, TEM is

i) Microscopic techniques for morphology very time consuming and needs to be performed on at characterization: Microscopic techniques such as least 100 nanosheets. Many works show TEM pictures atomic force microscopy (AFM) and transmission of 1-2 flakes, which may not be representative of the electron microscopy (TEM) are typically used for size whole material in dispersion. Furthermore, TEM can and thickness measurement of 2DMs. In case of AFM, damage the material during measurement due to the by depositing the nanosheets on a flat substrate, such use of highly energetic electron beams (typically of a as on silicon wafer, the morphology of individual few hundred kV acceleration voltages) at nanosheets can be directly measured in high-pressure vacuum. These microscopic techniques sub-nanometer scale resolution to obtain statistical are beneficial to study the morphology of 2D material analysis of size and thickness distribution of hybrids, for example, in the case of QDs or other nanosheets. It is recommended to scan different area nanomaterials deposited or grown directly on the

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2DM surface, due to their very high resolution. In this used for determining the theranostics effects – as case, it is crucial to determine the coverage of the such, we did not consider these features in the list of loaded drug or nanomaterial on the nanosheet, e.g., if characterization techniques. there are isolated NPs or aggregates, and if the Raman spectroscopy is one of the most popular coverage is similar for all flakes. A few studies have techniques to characterize graphene and other 2DMs used scanning electron microscopy (SEM) to [258]. It has been shown that Raman spectroscopy can characterize the nanosheets morphology, but the be used to identify graphene [259], to determine type resolution of this technique is not as good as that of and concentration of defects [260–262], amount and AFM or TEM. Thus, the smallest flakes cannot be type of doping [263] and many other properties. resolved. Furthermore, SEM requires conductive However, these studies have been conducted on a samples. In the case of non-conductive nanomaterials, high-quality graphene nanosheet, produced by deposition of a metallic film is required, sometimes mechanical exfoliation. Solution-processed or making more difficult to assess the morphology of chemically modified graphene materials, produced by individual flakes. different methods, do show very different Raman ii) Optical spectroscopic techniques: Several features because the spectrum (i.e. the intensity of the absorption/emission spectroscopies, such as Fourier peaks, their full width and half maximum, FWHM, Transform Infrared (FT-IR), UV-Visible, and Raman and positions) is strongly sensitive to the presence of spectroscopy are often used as a simple, non-invasive adsorbed or intercalated molecules and to technique to characterize the structure and changes in morphological changes, such as restacking of the chemical composition and/or degree of conjugation flakes, wrinkles, folding, defects, strain, and so on. in-situ. FT-IR is typically used to confirm Thus, the Raman analysis typically performed on functionalization with polyethylen glycol (PEG), perfect and clean graphene flakes produced by which has been used in many works to improve water mechanical exfoliation cannot be extended to solu- stability of the nanomaterial. In the case of graphene tion-processed graphene. For example, the FWHM of oxide, both FT-IR and UV-Vis can be used to confirm the 2D peak is used as a fingerprint to identify the reduction of the material: in the FT-IR spectrum, single-layer graphene (~25 cm-1) [259]– however, the the intensity of the peaks associated to the carbonyl, typical FWHM of the 2D peak of solution-processed carboxyl, hydroxyl and epoxy groups will decrease in graphene is around 50 cm-1. intensity. In the case of UV-Vis spectroscopy, the Despite these limitations, Raman spectroscopy absorption spectrum of graphene oxide shows a peak can still provide qualitative information on the at ~250 nm, due to π-π* transitions, which moves to thickness distribution of the flakes produced by slightly higher wavenumbers for reduced- liquid-phase exfoliation, as reported in [264–267], by graphene oxide (rGO). However, this analysis is only using a protocol based on the quality of the 2D peak qualitative and requires further characterization in fitting. Note, however that this method is very order to measure the C/O ratio. time-consuming as it requires analyzing 50-100 UV-Vis spectroscopy is also a potent technique isolated flakes, which could be hardly visible under for the structural characterization of semiconducting the optical microscope, and cannot be applied to GO 2DMs, such as TMDs, due to their unique exciton’s and rGO and solution-processed graphene produced features, which sharply change depending on defects by other methods. In the case of GO and rGO, these formation, functionalization or polymorph change. materials are defective, hence the Raman spectrum One can note that Li-intercalation has been used very sharply changes and therefore Raman spectroscopy often to produce TMDs dispersions: this method is cannot be used to identify the number of layers. well known to produce a substantial change in the Despite this limitation, Raman spectroscopy can still structure of the material, which changes from 2H to provide useful information regarding number of 1T polymorph, leading to metallic properties. This defects and to confirm reduction of GO. Defects fact should reflect in changes in the absorption analysis is typically done by using the intensity ratio spectrum and also changes in the photoluminescence between D and G peaks (I(D)/I(G)). However, this and fluorescence of the hybrid material, which is of analysis needs to be done with care: first, I(D)/I(G) fundamental importance in theranostics. Recent can be compared to other values reported in literature studies have also proposed protocols based on only if measurements are performed at the same absorption spectroscopy to identify size and thickness excitation wavelength, as this parameter strongly distribution of such materials [256]. It is noted that changes with the laser excitation [268]; second, UV-Vis spectroscopy is of fundamental importance in I(D)/I(G) do not always increase for increasing theranostics as it is used to confirm strong absorption defects concentration. Graphene shows a two-stage of light. Photoluminescence and fluorescence are also defects trajectory [262]: in Stage 1, starting from

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5468 pristine graphene, the Raman spectrum evolves as of oxidation of GO (decreasing 2θ value and peak follows: the D peak appears and I(D)/I(G) increases; broadening observed with increasing degree of the D', another defect activated peak located at ~1600 oxidation) [274] or confirmation of conjugation with cm-1, appears; all the peaks broaden and G and D' other materials. Note that XRD is a “bulk begin to overlap. In this stage, I(D)/I(G) can be used characterization technique”, providing average to estimate the amount of defects [269,270], while information on all material, while Raman I(D)/I(D’) can be used to distinguish between spectroscopy can be used on individual nanosheets. different type of defects [270]. At the end of Stage 1, iv) X-ray photoelectron spectroscopy (XPS): XPS I(D)/I(G) starts decreasing. As the number of defects is a surface-sensitive, quantitative spectroscopic keeps increasing, the Raman spectrum enters Stage 2, method that is often used to study the elemental and showing a marked decrease in the G peak position chemical composition of materials [275]. Oxygen and increase broadening of the peaks; I(D)/I(G) species of GO and rGO, and other elemental peaks sharply decreases towards zero and second-order from functionalization can be quantitatively peaks are no longer well defined. Thus, I(D)/I(G) characterized by XPS to estimate the degree of needs to be coupled with another Raman fit oxidation, reduction and/or functionalization [276]. parameter, such as the FWHM of the D, G or 2D peak, XPS can be a powerful technique to quantitatively in order to distinguish between the two-stages. Note determine the degree of oxidation or that GO belongs to stage 2- defective graphene, so functionalization, but the measurement should be reduction of the material should lead to a decrease of carefully carried out since it is highly surface-sensitive the peaks FWHM and to an increase in I(D)/(IG), technique (measurement depth is usually a few assuming rGO to remain in stage-2. nanometers). Moreover, possible contamination Similar mistakes done in the analysis of the during the sample preparation and/or measurement Raman spectrum of graphene can be seen also for should be carefully avoided, or possible adsorption of other 2DMs, where the Raman spectrum of a adventitious carbon from the atmosphere should be solution-processed nanosheet is compared to the taken into account during analysis. Finally, XPS Raman spectrum of a single-layer 2DM produced by cannot be made on individual flakes, so it requires mechanical exfoliation. Changes in FWHM and producing a film or a membrane. Thus, XPS provides positions of the peaks in a spectrum from average information on all material. A similar solution-processed material may not necessarily be technique is given by Energy-dispersive X-ray related to changes in thickness. spectroscopy (EDX, EDS or XEDS), which allows Because of the limits and challenges with optical analyzing much smaller material size, but needs to be spectroscopic techniques, these measurements should carried out in a scanning electron microscope. always be complemented with microscopic v) Thermogravimetric analysis (TGA) is often techniques. Note that since the resolution of optical used to determine the weight percentage of oxygen spectroscopic and microscopic techniques is very species, graphitic material, and/or degree of different, one should not expect to get exactly the conjugation with additional materials. same results. For example, if Raman spectroscopy vi) Zeta potential and Dynamic Light Scattering shows that the dispersion contains mostly single and (DLS): the zeta potential is the measure of surface few layers graphene, this should be confirmed by charge and potential distribution of a nanomaterial in TEM as well, although the numerical distribution may solution. In some cases where the particles are be slightly different. electrostatically stabilized in a colloidal suspension, It is pertinent to note that Raman spectroscopy, the zeta potential can be correlated to the being so sensitive to changes in the structure, is an sedimentation behavior [277]. For graphene-related ideal technique to investigate the changes in the materials, the zeta potential can be used to measure properties of the nanomaterial after each the degree of oxidation and functionalization, if the functionalization step. For example, if the Raman charge of the functionalized material is known. DLS is spectrum of the final vector does not show any G a simple and fast technique to measure the peak, this would possibly indicate the almost hydrodynamic size of nanomaterial dispersed in a complete disappearance of graphene. solution by using light scattering. Correlative iii) X-ray diffraction (XRD): XRD is often used relationship between the size measured by DLS and for qualitative identification of the crystal phases from microscopic techniques has been shown [278], but the study of the diffraction angle [271]. For graphene compared to microscopic techniques, DLS is a crude and other 2DMs, XRD is often used to measure the method to measure the size of 2DMs as the flakes will d-spacing between the layers (2θ = 26.3° for graphite be always crumpled into arbitrary shape in solution. (002) peak and 2θ = ~10 - 11° for GO) [272,273], degree Moreover, since DLS technique depends on the

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5469 intensity of scattering for size measurement, larger PTT and drug delivery (5%); other therapies (3%); particles can have more influence on the calculated imaging, PDT and drug delivery (2%); and imaging average than smaller particles, which can lead to and PDT (1%) (Figure 4C). From the analysis of the overestimation of size measurement, especially for literature concerning the different types of cancer solutions involving wide distribution of particle sizes, treated with GRMs as a theranostic tool, breast and such as in graphene dispersion [279]. cervical cancers appear the most studied (34% and 30% of the studies, respectively), followed by lung, 5. Comparing theranostic approaches and liver and brain cancer, which appeared results: a critical analysis approximatively in the same number of publications Through our review of the literature we found 84 (8%), and skin cancer (4%). Other types of cancer publications concerning graphene, GRMs, and (renal, bone, hepatic, Burkitt's lymphoma, ovarian, graphene hybrids (Figure 4A), which are summarized pancreatic, sarcoma, colon, prostate) represented the in Table 1, Table 2 and Table 3 according to the focus for less than the 2% of the papers (Figure 4D). In different type of combined applications, cancer, particular, even by looking at the different types of species of investigation, model as well as material and therapeutic approaches and the combinations of relative functionalization. Most of the cited studies strategies, the majority of the papers involving implied the use of imaging (MRI, X-ray CT, imaging and drug/gene delivery or PTT/PDT were fluorescent imaging, PAI, photothermal imaging, carried on breast and cervical cancer models, ultrasonography, and PET) as a diagnostic tool, independently from the type of approach (Figure 5). combined to one or more therapeutic strategies at the Focusing on the different molecules used for drug same time as shown in the Venn diagram (Figure 4B). delivery applications of GRMs for cancer theranostic, Focusing on the type of application, we found that, DOX emerged as a first-choice drug to functionalize among the different theranostic protocols, the as a common therapeutic drug (Figure 6). This fact combined use of imaging and drug or gene delivery, opens the window on the multitude of emerging new represents the majority of the analyzed studies (31%). drugs never conjugated with 2DMs, therefore, The second most commonly used method is offering the possibility of more innovative represented by combined imaging, drug delivery and functionalizations perhaps able to reduce possible PTT (24%); followed by combined imaging and PTT side effects and drug doses. (23%); imaging PTT and PDT (11%); imaging, PDT,

Figure 4. Analysis of the literature concerning GRMs. (A) The number of publications on graphene and GRMs in cancer theranostic (from 2008 to 2019). (B) Venn diagram based on the combination of imaging and the main therapeutic applications (drug delivery, PTT, and PDT) using GRMs as cancer nanotheranostic tools. (C) Histograms showing the numbers of publications concerning GRMs in cancer theranostics based on the different therapeutic approaches used in combination with imaging. (D) Overview on different types of cancer treated with GRMs as nanotheranostic tools (other types of cancer include: renal, bone, hepatic, Burkitt's lymphoma, ovarian, pancreatic, sarcoma, colon, and prostate).

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Figure 5. Percentages of various types of cancer treated with different combinations of theranostic approaches using GRMs. (A) Overview of different types of cancer treated with imaging and drug/gene delivery. (B) Overview of different types of cancer treated with imaging and PTT/PDT. (C) Overview of different types of cancer treated with imaging, drug/gene delivery, and PTT/PDT.

Figure 6. Number of publications related to the different drugs used for drug delivery based on GRMs, including targeting moieties.

Comparing the reports present in the literature conjugation of the material with PSs, genes, and drugs concerning the use of graphene for cancer for enhanced hyperthermia, gene therapy, and theranostics, GO emerges as a first-choice material for anticancer drug effectiveness. Therefore, not the design of nanotheranostic tools in 65% of the surprisingly, a good portion of the studies, where GO studies, followed by GQDs (14%), and rGO (12%), or NGO have been exploited for imaging application, while only 9% of the tested materials included is associated with drug/gene delivery alone or in graphene or other graphene hybrids (Figure 7A). This combination with PDT/PTT (Figure 7B). view is mainly due to GO superior biocompatibility, However, rGO resulted in one of the most the ease of functionalization, due to the considerable effective materials in terms of tumor reduction ability, presence of chemical polar groups, as well as high demonstrating to be able to totally ablate the tumor in dispersibility in aqueous biological environments and vivo [281]. This seems to be due to its physicochemical prolonged blood circulation time compared to other properties, such as the strong NIR absorbance GRMs [280]. In particular, the high surface-to-volume capability compared to GO and other GRMs, ratio allows the conjugation with different molecules endowing it with a high photothermal conversion able to improve and expand the range and efficiency for cancer thermal ablation [282]. The effectiveness of simultaneous imaging methods and number of publications focusing on the use of rGO for multiple therapies applicable in the same imaging application associated to PDT /PTT, alone or nanotheranostic tool. Indeed, the diagnostic and in combination with drug/gene delivery, is much therapeutic capability can be improved by the higher compared to the use of rGO for imaging and

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5471 drug/gene delivery alone (Figure 7B). Indeed, in taken into account given future clinical translation, in several studies, rGO has shown to be an appealing accordance with the different type of cancer to be candidate for controlled irradiation-responsive treated. In general, promising results were obtained nanoplatforms. Moreover, its therapeutic properties by both in vitro and in vivo studies, however, animal can be enhanced thanks to the association with models showed a more variable response, obtaining a noninvasive temperature-dependent drug release in total cancer eradication only in 20 treatments response to illumination or pH-depended drug [74,80,84,87–90,93–97,123,281,286–291] out of 59. Of release in response to acidic conditions similar to these successful studies, only two were carried out those of cancer cells [51,283]. The ability of rGO to only using gene/drug delivery while all the others absorb NIR light and convert it into heat can also be exploited PTT or PDT alone or in combination with exploited for PAI, due to the consequent ultrasonic gene/drug delivery. A summary of the advantages emission, resulting in a theranostic nanoplatform with and disadvantages of GRMs for cancer theranostic dual-enhanced photothermal conversion properties applications emerged from the reported studies is [89,114]. In addition, rGO displayed magnetic illustrated in Table 6. properties and was able to improve the contrast in Concerning the analysis of 2DM beyond both MRI and X-ray CT [284]. This scenario, is of graphene, it is clear that they are rising increasing particular interest in theranostics, since MRI interest; as shown by the recently growing number of represents the most powerful imaging method for publications based on these promising tools for cancer tumor diagnosis and early detection [285]. theranostics (Figure 8A). Considering GQDs, they were mainly exploited As shown in Figure 8B these materials were for imaging properties, due to their intrinsic mainly employed for imaging in association with photoluminescence, making them ideal candidates for approaches involving PTT alone (41%) or in imaging purpose, but were also used in conjugated combination with drug/gene delivery (22%), and forms for drug/gene delivery (Figure 7B). However, PDT (19%). By the analysis of the literature better results with GQDs in terms of cancer ablation concerning the different types of cancer treated with seems to be obtained through the association of 2DMs beyond graphene as a theranostic tool, breast imaging with PTT or PDT, alone or in combination, and liver cancers appear the most studied (42 and 24% leading to considerable (53.4%) [83] or even total of the studies, respectively), followed by cervical [97,171] tumor eradication. However, the effects (18%), lung (7%), brain (3%), while only a small showed different potency also dependently on the portion of works involved colorectal, skin and bone different cell ability to internalize the materials and cancer, which appeared approximatively in the same their susceptibility to reactive oxygen species [121]. number of publications (2%) (Figure 8C). These results suggest that these aspects should be

Figure 7. Analysis of the literature concerning the type of GRMs. (A) Percentages of the different types of GRMs used in theranostics. (B) Overview of different types of GRMs and their theranostic application.

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Figure 8. Analysis of the literature concerning 2DMs beyond graphene. (A) The number of publications of 2DMs in cancer theranostics. (B) The number of publications concerning 2DMs in cancer theranostics based on the different therapeutic approaches used in combination with imaging. (C) Overview of different types of cancer treated with 2DMs.

Table 6. Table showing the main advantages and disadvantages of GRMs for cancer theranostics applications (Y=yes, N=no).

Property GO rGO Graphene Other 2DMs High surface area Y Y Y Y Defective Y, high Y, low N Depends on the 2D material Functionalization Very easy Easy Possible, but more difficult than GO or rGO Some functionalization routes existing, depending on the 2D material Dispersibility in water Y N N No, with the exception of few 2D materials (e.g. MXene) Stability in air Y Y Y Some 2D (e.g. phosphorene has limited stability in air) Electronic properties Generally small conductivity, but Conductive Highly conductive Metallic, semiconducting or insulating, depending on the 2D depends on the degree of oxidation. material NIR Absorption Good Very good Good Depends on the 2D material Photoluminescence Y, weak N N some of the 2D materials (e.g. TMDs) show photoluminescence Biocompatibility Depends on the way of production, physicochemical properties and doses

Although the applications of new emerging extinction coefficient of 25.2 L g−1 cm−1, compared to 2DMs are still rare in the field of cancer theranostics that of GO nanosheets (3.6 L g−1 cm−1) [294]. However, and biomedicine in general, from the works here their relatively low photothermal conversion analyzed it is possible to comment on the merits and efficiency could limit their future applications. The potential clinical translation of those new materias. development of Ta4C3 allowed to obtain both optimal Among new emerging 2DMs, antimonene QDs and photoabsorpion properties and photothermal GDY exhibit a photothermal conversion efficacy (45.5 conversion efficiency (44.7%) thanks to the strong and 42%, respectively) higher than traditional 2DMs, absorption band, similar to those of other such as graphene, GO, MoS2, and BP [225]. This conventional 2DMs, such as graphene [110] and MoS2 property makes these emerging nanomaterials [152]. particularly suitable for PTT. GDY has also shown to Compared to other types of 2DMs, such as MoS2 exceed pristine graphene nanosheets in terms of drug [147], BP [295], graphene [110,294], Ti3C2, and Ta4C3 loading ability, with a DOX loading content equal to [296], Nb2C exhibits a strong and almost constant 38% [292], due to its structure, composed by both sp- optical absorption in the NIR I and NIR II windows. and sp2-hybridized carbon atoms, allowing GDY to The possibility of employing PTA with high preserve its conjugated structure even after covalent conversion efficiency also in the NIR II window functionalization. This ability shows promises in the (1000-1300 nm), such as Nb2C, is expected to attract field of nanotheranostic, since it could allow increasing interest in the future of cancer therapy, simultaneous covalent link of several molecules, allowing a higher tissue penetration depth. including drugs and targeting, tracking or bioactive It is clear that the physicochemical properties of structures. 2DMs are urging their use in biomedicine [297]. For MXenes in general possess remarkable instance, thanks to the higher water dispersibility and photothermal performance [194,293]. Ti3C2 colloidal stability, g-C3N4 and BP are supposed to be nanosheets are characterized by an exceptional high more advanced in the biomedical field, without the

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5473 use of any surfactants. In contrast, despite their new 2DMs beyond graphene) used in cancer limited stability in aqueous dispersions [298], theranostics. On the whole, by an analysis of the electrically inert h-BN sheets seem to be more imaging approaches, the most used imaging biocompatible and better suited for the drug delivery. technique was CLSM (42 papers), followed by With the aim to improve the biocompatibility of 2DMs fluorescence imaging (41 papers), MRI (37 papers), as well as to control their solubility and and PAI (28 papers), while other imaging methods biodistribution, the development of new covalent were less frequently used (Figure 10). As mentioned functionalizations will be an interesting effort for before, even if in several studies, the fluorescence future studies. Moreover, following this path, the properties are claimed as possible diagnosis syntheses for theranostic applications needs to focus strategies, it is important to mention that fluorescence on different parameters that could dramatically imaging has not entered the clinic yet. Some change the fate of the new synthetized 2DMs: outstanding examples are reported in Figure 11 which dimensions, surface functionalities, and aqueous summarizes some of the most complex and dispersibility. multimodal applications of these nanotools available Overall, Figure 9 shows a comparison among the to date. percentage of different type of materials (GRMs and

Figure 9. Graphic representation of the discussed works concerning the use of GRMs and other 2DMs in cancer theranostics. “Other 2D materials” includes: graphdiyne, hexagonal boron nitride, silicene, antimonene, germanene, biotite, metal organic frameworks, and layered double hydroxide.

Figure 10. Overview of different types of imaging modalities used for theranostic purposes (GRMs and 2DMs). Abbreviations: CLSM = Confocal Laser Scanning Microscopy, CT imaging = Computed Tomography Imaging, FLIM = Fluorescence-lifetime imaging microscopy, MRI = Magnetic Resonance Imaging, NIR imaging = Near-Infrared Imaging, PAI = Photoacoustic Imaging, PAT = Photoacoustic Tomography, PET = Positron-emission tomography, UCL = Laser Scanning Upconversion Luminescence Imaging, SERS imaging = Surface Enhanced Raman Scattering, SPECT = Single-photon emission computed tomography, US imaging = Ultrasound imaging.

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Figure 11. Examples of GRM-based imaging and therapy for combined and multimodal applications in cancer theranostic. (A) Schematic illustration of drug delivery theranostic strategy based on GQD fluorescence for a programmatic monitoring of the anticancer drug delivery, release, and response. Adapted with permission from [56], copyright 2017 American Chemical Society. (B) Theranostic application of GQDs fluorescence; NIR fluorescence imaging of tumor-bearing mice intravenously injected with free IR780 or IR780/GQDs-FA up to 48 h. Adapted with permission from [288], copyright 2017 American Chemical Society. (C) Tumor photographs, histological images, tumor volume and body weight of mice trated with GO-based nanotheranostic tool. Adapted with permission from [85], copyright 2018 American Chemical Society.

concerns non-specific targeting, which is especially 6. Conclusions: how to develop 2D relevant for drugs with a broad range of targets that materials for theranostics could lead to severe organs damage [16]. On the other hand, the delivery of therapeutic molecules could be 6.1. Targeting and resolution: two historical difficult for those drugs that can hardly penetrate limits to consider tumor cells through the blood or showing a short The recent advances in new 2DM development circulation half-live [299]. were directed to overcome some of the persisting The delivery of drugs at the requested site by limitations of conventional cancer-fight modalities nanomaterials is still challenging, even for FDA such as: i) the nonspecific targeting and ii) low approved particles. Notably, it has been determined resolution in imaging and difficulties in diagnosis. that less than 1% of NPs explored for anticancer drug i) The nonspecific targeting. One of the main delivery typically reach the tumor [300]. It is far too limitations of current chemotherapeutic drugs early to conclude if 2DMs have a chance of entering

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5475 the clinic for drug delivery, and eventually to perform quenching effects. However, while some of the better than current clinically approved nanomedicines imaging opportunities offered by nanomaterials [301–304]. Undoubtedly, the high surface-to-volume appear to be more easily and readily applicable to the ratio makes 2DMs very useful in this sense allowing a clinic, such as the implementation of MRI- and CT high load of drugs and the functionalization with imaging-based technologies, which interact mainly at specific ligands recognized by particular receptors the atomic level allowing the visual inspection at the overexpressed on specific tumor cells, facilitating the organ length scale, other optical techniques selective targeted process of internalization and the exploitable at the molecular level by these delivery of chemotherapeutic agents or genes up to nanosystems (e.g. fluorescence-based imaging for the action site. Furthermore, the multiple diagnosis and guided therapy) face hurdles for functionalizations are not inside of the particles as it bench-to-bedside translation. Indeed, for instance, can happen with multistage vectors [305]. The drugs fluorescence imaging is being more an experimental in the case of 2DMs may have an easier way of release. approach and requires the development of new However, on the other hand, multistage anticancer technologies before adoption into the clinic. Indeed, vectors can incorporate several components, these despite the in vivo promising results, optical imaging components are activated sequentially in order to is usually tested in mouse xenograft models where the successively address transport barriers; this tumors are usually located superficially and therefore “multistage advantage” has not been reported yet for easily detectable. Moreover, fluorescent molecular 2DMs. Microenvironmental priming strategies in the imaging is affected by the biological tissue theranostics context of 2DMs are highly desired. autofluorescence, photobleaching of fluorescent dyes, Moreover, a polymer coating would allow the carrier and significant spectra overlap between broadband to circulate unnoticed towards the immune system, molecules [311–313]. In general, the issue could be while the cargo release could be programmed more easily overcome by the design of multi-modal according to the presence of various stimuli, such as nanoplatform integrating both a new optical modality pH and temperature variations, that typically occur and an already proven clinical methodology to extend into the cancer tissues. To overcome the issue its utility, aimed at improving and assisting a concerning the lack of target specificity, further standard methodology rather than disrupt the information concerning the biodistribution of common clinical workflow. One more point to nanomaterials in the context of cancer theranostic consider is the fact that nanomedicine development research are also still needed. Indeed, toxicological has allowed rethinking the concept of studies concerning GRM biodistribution investigation nanotheranostics, expanding its notion beyond the have highlighted the impact of critical aspects on classical meaning of physical entity that affords both nanomaterial fate into the body, including diagnostic and therapeutic functions to a broad physicochemical properties, such as dimension and generic nano-enabled approach. In this view, a new functionalization, as well as the formation of protein theranostic concept is exploiting the molecular corona [306–309]. Moreover, a key aspect still not imaging functionalities not only for diagnosis but also considered in the studies analyzed here and that to aid or guide a nanotherapeutic procedure [9]. It is could influence the biodistribution of nanomaterials, therefore noteworthy that in some cases fluorescence is the difference between healthy and cancer blood can be used as optical imaging to aid or guide vessels [310]. Indeed, the latter is characterized by minimally invasive surgery, such as in the case of slower blood flow and the presence of holes brain tumors [314] or for the detection of sentinel (fenestration) that should be taken into consideration lymph nodes [315]. in 2D design and production to predict the possible Concerning other imaging techniques, while extravasation to reach the tumor region [302]. radiolabeling-based nuclear imaging has been ii) Low resolution in imaging and difficulties in explored for 2DMs thanks to superior sensitivity and the diagnosis. The low-resolution performance in the possibility to obtain whole-body images imaging represents one more hurdle for cancer compared to fluorescent labeling, there is still a lack of theranostics development. The suitability of the studies investigating nanomaterials for ultrasono- promising 2DMs here discussed to serve as imaging graphy, an extreme safe imaging modality present in tools for cancer nanotheranostic purpose mainly relies the every-day clinical practice [316]. on their outstanding physicochemical characteristics The cardinal feature that renders 2DMs more useful for cancer diagnosis and guided therapy, as suitable compared to other particles is undoubtedly reported in all the studies analyzed. For example, GO their multiple imaging advantages, already proven by exhibit fluorescence from the visible to NIR range and the analysis of the scientific literature [108,317]. The NIR fluorescent dyes to reduce the intrinsic graphene multiple imaging should, however, take into

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5476 consideration the real cancer needs, the real different reviews have been reported to evaluate this translation into the clinic when it will come to the interaction [280,321–328]. Indeed, the immune system diagnosis of tumors in human patients, and the governs every aspect of our health and plays a crucial potential differences among mice and humans in role in the response of the organism to tumor terms of depth of tissues and, therefore, also in terms eradication as well as to cancer therapy. In this view, of sensitivity. Finally, for imaging as well as for the immunotherapy is able to trigger the immune system drug delivery aims, it is vital to analyze and, possibly, of cancer patients to elicit a strong antitumor quantify the material distribution in each type of response. In view of their multifunctional properties, theranostic study, taking into account the different new 2DMs can serve as nanotheranostic tools for cancer models, since the distribution may vary cancer immunotherapy thanks to their ability to depending also on the cancer tissues. modulate the immune system to eradicate cancer. Moreover, their functionalization with specific 6.2. Necessary considerations before the design monoclonal antibodies, such as Rituxan, allows to of nanomaterial theranostics specifically recognizing the cancer cells leading to The first and most crucial aspect still requiring to tumor destruction [49]. The potentialities of 2DMs as be fully elucidated to develop and design theranostic immunotherapeutics need further investigations as tools, allowing their translational application into the done for other types of nanomaterials [329]. clinic, are the assessment of their specific toxicity. In So far, in vivo oncological nanotheranostic this view, their toxicological effects and the research has been performed primarily in mice underlying mechanisms after entering the organism models, which are often not sufficiently adequate (both in cancer and healthy model at the same time clinically relevant models. In these models, the tumor and with the same material) need further usually derives from immortalized human tumor cell investigation, in particular following intravenous lines after subcutaneous injection, which shows a injection, representing the main route of variable success rate of cancer xenograft growth and administration of nanomaterials in nanotheranostics not well represent the patient tumor heterogeneity [318]. offered by patient-derived xenograft models or The understanding of the larger picture accurate genetically engineered mouse models. To depicting the relationship between the structure and accelerate the advance to clinical implementation, the activity of the materials, from living systems to the adequate supportive data related at least to nanotool molecular level, can be ensured through new systems performance in terms of imaging, along with biology approaches [319], designing robust and long-term assessment of their safety, stability and validated experimental methods [320]. biodistribution, need to be acquired in larger animal The relationship mentioned above can be models different from mice, which share more extended to the application level, designing the physiologic similarities with humans and present a structure to modulate different physicochemical longer life span, such as swine and non-human properties and potential biological impacts suitable primates. In particular, these translational for specific imaging and therapeutic procedures in (preclinical) models (to human clinical trials) would consideration of the specific tissue and form of cancer provide a suitable step to evaluate the long-term taken into consideration. This correlation is not destination of theranostic nanotools in the body, straightforward, and its clarification requires a which is particularly relevant for slowly degradable multi-interdisciplinary approach, involving clinicians or nondegradable nanomaterials, to assess whether and scientists whose efforts rely on the field of and to what extent they can persist and accumulate in material science, chemistry, physics, biology, and the organism for a long time, eventually leading to toxicology. However, it must be considered that the chronic inflammation. The possibility to control these toxicological profile of nanomaterials presents Janus’s aspects would allow mitigating the side effects and double-face in the cancer fight. On the one hand, the therefore improving the therapeutic efficacy. potential systemic toxicity may cause harm. On the As a critical step for the evaluation of their other hand, localized toxic effects can be useful for biocompatibility, the investigation of the in vivo clinically controlled cancer ablation, when the impact of 2DMs applied to clinical application mechanism of action is known. requires further toxicity and biodistribution analysis The investigation of the toxicity should not as well as metabolism studies that should be carried overlook their impact on the immune system, since out applying the principles of reduction, refinement, nanomaterials directly interact with the blood and replacement. This long trial needs to be immune cells when entering the bloodstream during a accomplished according to specific standardized medical procedure. In this view, several studies and procedures at an international level, such as the

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5477 regulatory European guidelines EN ISO 10993. (e.g., lateral size, number of layers, shape, surface Among these tests, the evaluation of irritation charge, elasticity, chemical modification, etc.) [334]. and sensitization reactions would be critical to Indeed, it has been highlighted the importance of determine the allergic response and the localized considering the family of GRMs as composed by inflammatory response after, for instance, skin different individual materials with specific contact. This aspect is particularly relevant for topical physicochemical characteristics, which in turn will application of biomedical nanotool, like PSs applied to affect their toxicity [253], and the same concept should the skin or nanosystems for wound healing [330]. be applied for their theranostic potentialities. Moreover, beside the assessment of systemic toxicity Moreover, a rational, systematic classification, following a single or multiple dose during a short allowing a scientific comparison among the different period (acute toxicity), it is crucial to evaluate the materials and their specific characteristics, is required impact of repeated doses for a prolonged period to optimize and predict their possible theranostic (subacute, subchronic, and chronic systemic toxicity), applications. The current Food and Drug since the latter represents the most common Administration (FDA) process to approve nanodrugs administration of 2DM-based medical devices. does not differ from that of any other drug or biologic Mutagenicity, carcinogenicity, reproductive and [16,335–337], involving three phases of clinical trials, development toxicity also need to be investigated, but a specific regulation of nanotechnology medical especially for materials destined to have a long-term products is still missing and expected to be released exposure to the human body. In case of materials [336]. To this end, it is essential to fully elucidate the applied for therapy, such as in the case of cancer biocompatibility of nanomaterials to draw theranostics, it would be important to investigate the conclusions on their toxicity and possible hazards in biocompatibility in healthy tissues by using order to develop proper guidelines. experimental model developed to mimic pathological Altogether, these observations show a puzzling conditions of the surrounded organs/tissues, since picture where many aspects still need to be further the outcome of the nano-therapy will be influenced by explored or improved. However, the growing the morbid condition [331]. research in the field highlights how the scientific The development of scalable nanotools is also a community is becoming aware that cooperation and relevant need for their clinical translation. There are multi- and inter-disciplinary approaches are essential still critical issues related to the large-scale production to address the abovementioned limits in designing of graphene, GRMs, graphene hybrids, and, even cancer theranostics. We hope that our conclusions will more, for that of the other 2DMs here discussed. These help the community to reflect on the key limits are mainly represented by the difficulties in considerations and open questions for 2D theranostics obtaining a large number of high-quality products, development. Overall, it is clear that the applications dispersible in physiological solutions and at high of graphene, graphene hybrids, and other novel 2DMs concentrations, in a simple, low-budget, biocom- for cancer theranostics have the potentialities to offer patible, sterile and green-environmentally friendly a substantial contribution in the fight against cancer. manner. Indeed, for any nanomaterial toxicological evaluation and the subsequent medical application, 6.3. The future of 2D materials: clinical there is a requirement to avoid chemical and translation biological impurities (e.g., endotoxin contamination) 2D materials represent a thriving field not least [332,333], which can derive from residual reagents in cancer theranostics, due to the manifold advantages used for their synthesis and the lack of sterile of these materials including their varied conditions during their production, respectively. physicochemical properties, and low-cost production. Therefore, in studies concerning the investigation of The challenge of translating graphene and other theranostic tools, including the new 2DMs here 2DMs into the clinic is not a new undertaking. Similar presented, these aspects must be taken into concerns exist in every cutting-edge technology in the consideration. Indeed, all the above-mentioned biomedical field, such as gene therapy, genome requirements are not easy and obvious to obtain for editing or stem cell therapeutics. In recent years, we all the materials here analyzed for cancer theranostics. have witnessed an exceptional accumulation of new In addition, defects and variability in size and knowledge on graphene and its applications in thickness that can occur during production may affect medicine. In terms of nanotechnologies applied to the performance and the theranostic effectiveness of biology, graphene is represented by two different the material. It is crucial to deeply characterize the forms: as an engineered graphene-incorporating materials through well-established and standardized device, for use as sensors and implants, or as a highly techniques to evaluate the impact of their properties oxygenated and structurally defective form of

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5478 graphene oxide and its derivatives, stable in water the accompanying figures. All co-authors approved and easy to functionalize. The latter formulations are the final version of the present manuscript. currently explored in different drug delivery, photothermal, photodynamic, pharmacological, Competing Interests toxicological, and theranostical studies. The growing The authors have declared that no competing interest in the translation of graphene and other 2DMs interest exists. into a range of application areas, including medicine, promoted, in part, by the Graphene Flagship project References [2], is thus fueling great expectations. Graphene has 1. Novoselov KS, Fal′ko VI, Colombo L, Gellert PR, Schwab MG, Kim K. A been tested in vitro and in vivo obtaining promising roadmap for graphene. Nature. 2012; 490: 192–200. 2. Ferrari AC, Bonaccorso F, Fal’ko V, et al. 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324. Orecchioni M, Jasim DA, Pescatori M, et al. Molecular and Genomic Commission (success rate about 10%), the “200 Young Impact of Large and Small Lateral Dimension Graphene Oxide Sheets on Human Immune Cells from Healthy Donors. Advanced Healthcare Best Talents of Italy 2011” by the Italian Ministry of Materials. 2016; 5: 276–87. Youth, and “Bedside to bench & Back Award” by the 325. Russier J, León V, Orecchioni M, et al. Few-Layer Graphene Kills Selectively Tumor Cells from Myelomonocytic Leukemia Patients. National Institutes of Health, Bethesda, USA. She has Angew Chem Int Ed Engl. 2017; 56: 3014–9. been the Scientific Coordinator of two 326. Orecchioni M, Ménard-Moyon C, Delogu LG, Bianco A. Graphene and interdisciplinary European Projects under horizon the immune system: Challenges and potentiality. Adv Drug Deliv Rev. 2016; 105: 163–75. 2020 program on nanomedicine and nanomaterials 327. Avitabile E, Bedognetti D, Ciofani G, Bianco A, Delogu LG. How can immune interactions (2 M euros) . Her research nanotechnology help the fight against breast cancer? Nanoscale. 2018; 10: 11719–31. focuses on immune/bio interaction and safety of 328. Keshavan S, Calligari P, Stella L, Fusco L, Delogu LG, Fadeel B. Nano-bio carbon-based nanomaterials as well as on the interactions: a neutrophil-centric view. Cell Death Dis. 2019; 10: 569. 329. Song W, Musetti SN, Huang L. Nanomaterials for cancer development of biomedical applications for new immunotherapy. Biomaterials. 2017; 148: 16–30. carbon-based and 2D materials. 330. Li J, Zhou C, Luo C, et al. N-acetyl cysteine-loaded graphene oxide-collagen hybrid membrane for scarless wound healing. Theranostics. 2019; 9: 5839–53. 331. Giardino R, Fini M, Aldini N, Parrilli A. Testing the in vivo biocompatibility of biocomposites. Biomedical Composites. 2009; 385– 410. 332. Mukherjee SP, Kostarelos K, Fadeel B. Cytokine Profiling of Primary Human Macrophages Exposed to Endotoxin-Free Graphene Oxide: Size-Independent NLRP3 Inflammasome Activation. Adv Healthc Mater. 2018; 7: 1700815. 333. Li Y, Boraschi D. Endotoxin contamination: a key element in the interpretation of nanosafety studies. Nanomedicine (Lond). 2016; 11: 269–87. 334. Gao X, Lowry GV. Progress towards standardized and validated characterizations for measuring physicochemical properties of Prof. Cinzia Casiraghi Helds manufactured nanomaterials relevant to nano health and safety risks. NanoImpact. 2018; 9: 14–30. a Chair in Nanoscience at the Department of 335. Bobo D, Robinson KJ, Islam J, Thurecht KJ, Corrie SR. Chemistry, University of Manchester (UK). She Nanoparticle-Based Medicines: A Review of FDA-Approved Materials and Clinical Trials to Date. Pharm Res. 2016; 33: 2373–87. received her B.Sc. and M.Sc. in Nuclear Engineering 336. Havel H, Finch G, Strode P, et al. Nanomedicines: From Bench to Bedside from Politecnico di Milano (Italy) and her Ph.D. in and Beyond. AAPS J. 2016; 18: 1373–8. Electrical Engineering from the University of 337. Sainz V, Conniot J, Matos AI, et al. Regulatory aspects on nanomedicines. Biochem Biophys Res Commun. 2015; 468: 504–10. Cambridge (UK). In 2005, she was awarded with an 338. Kong W, Kum H, Bae S-H, et al. Path towards graphene Oppenheimer Early Career Research Fellowship, commercialization from lab to market. Nat Nanotechnol. 2019; 14: 927– 38. followed by the Humboldt Research Fellowship and the prestigious Kovalevskaja Award (1.5M Euro). Her Author Biographies current research work is focused on the development of biocompatible 2D inks and their use in printed electronics and biomedical applications. She is leading expert on Raman spectroscopy, used to characterize a wide range of carbon-based nanomaterials. She is recipient of the Leverhulme Award in Engineering (2016), the Marlow Award (2014), given by the Royal Society of Chemistry in recognition of her work on Raman spectroscopy, and an ERC Consolidator grant (2015).

Dr. Lucia Gemma Delogu is Assistant Professor of Biochemistry at the University of Padua (Padua, Italy) (www.delogulab.eu). She received her Ph.D. in Biochemistry in 2008. She integrated her Ph.D. research at the Sanford-Burnham Institute (La Jolla San Diego, USA) and then was postdoctoral fellow at the University of Southern California (Los Angeles, USA). She has worked as Visiting Professor at the University of Dresden, Germany (2016-2017). She has received several Prof. Bengt Fadeel is a awards, including the “Marie S. Curie Individual Full Professor of Medical Inflammation Research at Fellow” under Horizon 2020 by the European Karolinska Institutet, Stockholm, Sweden, since 2010,

http://www.thno.org Theranostics 2020, Vol. 10, Issue 12 5486 and Head of the Division of Molecular Toxicology at (Italy) studying the toxicological effects of graphene. the Institute of Environmental Medicine at Karolinska She is currently a Ph.D. student in Nanotechnology at Institutet since 2009. He received his M.D. and Ph.D. the same university (Prof. M. Prato), where she is from Karolinska Institutet in 1997 and 1999, integrating her research as a Visiting Ph.D. student at respectively. He is a Fellow of the Academy of the Italian Institute of Technology and the University Toxicological Sciences. Dr. Fadeel has been engaged of Genoa (Italy). Her research is centered on the in several EU-funded nanosafety projects as he is immunocompatibility and biomedical applications of currently a member of the 10-year GRAPHENE new nanocarbons. Flagship Project, focusing on safety assessment of graphene-based materials. His research interests span from cell death signaling to mechanisms of inflammation to toxicological investigations of engineered nanomaterials as well as medical applications of nanomaterials. He was awarded the national Environmental Medicine Prize in 2011 for his research on the opportunities and risks of the emerging nanotechnologies.

Dr. Guotao Peng received her B.Sc. in Environmental Science from Sichuan University (China). She obtained her Ph.D. in 2017 at Fudan University (China) focusing on the mechanistic understanding of toxic cyanobacterial blooms, integrating her research at University of Tennessee (USA) as a visiting Ph.D. student. She then worked as a postdoctoral fellow at Tongji University (China) focusing on environmental health and safety aspects Dr. Laura Fusco received her of nanomaterials. Currently, she is a postdoctoral B.Sc. in Medical Biotechnologies from the University fellow at Karolinska Institutet in Stockholm in the of Milan (Italy) studying the bioeffects of ambient frame of the GRAPHENE Flagship project; she particulate matter. After receiving her M.Sc. in focuses on immune interactions of graphene-related Medical Biotechnologies and Molecular Medicine materials using in vitro and in vivo models. from the University of Trieste (Italy), in 2018 she earned her Ph.D. in Chemistry from the same university (Prof. M. Prato), with a project on the toxicological effects of graphene at the skin level, supported by the European Union H2020 Programme, integrating her research at the Karolinska Institutet (Prof. B. Fadeel) as a visiting Ph.D. student. Currently, she is a Postdoctoral Fellow at the University of Trieste and a Visiting Scientist at Sidra Medicine (Qatar) in the framework of a H2020-MSCA-RISE Dr. Yuyoung Shin project concerning the immune activity of carbon obtained her B.Sc. Honours degree in Chemistry from nanomaterials. University of Sussex (UK) in 2010, then completed an MPhil in Chemistry from University of Cambridge in 2012. Following this, she undertook her Ph.D. in chemistry funded by the EPSRC, with Prof Cinzia Casiraghi at University of Manchester (UK), working on synthesis and characterisation of graphene-based membranes. After the completion of her Ph.D. in 2017, she joined the 2D-Health project as a post-doctoral research associate focusing on synthesis of graphene and other 2D crystal inks for biomedical imaging applications. Arianna Gazzi received her M.Sc. in Pharmacy from the University of Trieste

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host-tumor interactions, and to understand its relationship with treatment effectiveness.

Dr. Sandra Vranic obtained her B.Sc. Degree in Molecular Biology and Physiology at the University of Belgrade (Serbia) in Dr. Flavia Vitale is an 2007. After graduation, she completed MRes Degree Assitant Professor in the Departments of Neurology, in Toxicology at University Paris Diderot – Paris 7 Bioengineering and Physical Medicine and (France), earning her Ph.D. in Toxicology from the Rehabilitation at the University of Pennsylvania same university. She focused on interactions of (USA). She received her Ph.D. in Chemical engineered nanoparticles with cells, especially on the Engineering at the Università di Roma "La Sapienza" mechanisms of their internalization and subsequent (Italy), then completed her training in the Department cellular effects. After her Ph.D., she obtained Japanese of Chemical and Biomolecular Engineering a at Rice Society for Promotion of Science (JSPS) postdoctoral University (USA) in 2015, where she was a R. A. fellowship at Nagoya University and Tokyo Welch Foundation Postdoctoral Fellow. Dr. Vitale has University of Science in Japan, where she studied the received a number of awards, including the Graduate effects of silica nanoparticles in mice and Zebra fish. Research Fellowship, University of Rome "La In 2015, she joined the Nanomedicine Lab as a Marie Sapienza", the Taking Flight Award from CURE and Curie Research Fellow under the RADDEL ITN the McCabe Fellow Award. Dr. Vitale research project studying nanocapsules filled with radiometals focuses on application of carbon nanostructures and aimed for biomedical applications in the area of MXene to bioelectronic interfaces and biosensors. cancer diagnosis and therapy. She worked as a lead scientist in Graphene Flagship and 2D Health projects, and currently acts as a PI of EU funded Horizon 2020 project BIORIMA. In 2018, she was appointed as Lecturer in Nano-Cell Biology. Her team aims to investigate cellular and molecular biology of graphene and other 2D materials in order to discover new therapeutic applications of these materials.

Dr. Acelya Yilmazer is an Assistant Professor at the Department of Biomedical Engineering in Ankara University (Turkey) since 2015. In 2012, she obtained her Ph.D. in the Nanomedicine Lab based in the School of Pharmacy, University College London (Prof. K. Kostarelos). Dr. Davide Bedognetti is Later, she continued her postdoctoral work in the the Director of the Cancer Program at Sidra Medicine same lab on in vivo cell reprogramming. She has (Qatar). Dr. Bedognetti received his M.D. and Ph.D. in extensive experience on in vitro and in vivo preclinical Clinical and Experimental Oncology and Hematology evaluation of nanomaterials. Her research group is from the University of Genoa (Italy). After completing focusing on the use of nanomaterials and cell his medical residency in Medical Oncology, he joined reprogramming technologies in cancer or the Infectious Disease and Immunogenetics Section regenerative medicine. She is coordinating the (IDIS) of the US National Institutes of Health (NIH) genotoxicity related work package of the Graphene where he completed his post-doctoral fellowship. Dr. Flagera project "G-IMMUNOMICS". She has been Bedognetti’s team employs high-throughput selected as the “Best Young Investigator” in Turkey approaches to de-convolute the molecular network of by the Turkish Society of Medical Biology and Genetics in 2013 and given the “Young Scientist Award” by the Turkish Academy of Sciences in 2019.

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Prof. Xinliang Feng is a full professor and the head of the Chair of Molecular Functional Materials at Technische Universität Dresden. He has published more than 450 research articles which have attracted more than 51000 citations with H-index of 108 (Google Scholar). He has been awarded several prestigious prizes such as IUPAC Prize for Young Chemists (2009), European Research Council (ERC) Starting Grant Award (2012), Journal of Materials Chemistry Lectureship Award (2013), ChemComm Emerging Investigator Lectureship (2014), Fellow of the Royal Society of Chemistry (FRSC, 2014), Highly Cited Researcher (Thomson Reuters, 2014-2018), Small Young Innovator Award (2017), Hamburg Science Award (2017), EU-40 Materials Prize (2018), ERC Consolidator Grant Award (2018), member of the European Academy of Sciences (2019) and member of the Academia Europaea (2019). He is an Advisory Board Member for Advanced Materials, Chemical Science, Journal of Materials Chemistry A, ChemNanoMat, Energy Storage Materials, Small Methods, Chemistry -An Asian Journal, Trends in Chemistry, etc. He is the Head of ESF Young Research Group "Graphene Center Dresden", and Working Package Leader of WP Functional Foams & Coatings for European Commission’s pilot project “Graphene Flagship”.

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